Cationic dry powders

ABSTRACT

The invention relates to respirable dry particles that contain one or more divalent metal cations, such as calcium, in an amount of less than 3% by weight, and to dry powders that contain the respirable particles. The dry particles can further contain an active agent, or can be used as carrier particles to deliver an active agent.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/387,855, filed on Sep. 29, 2010 and PCT/US11/49435, filed on Aug. 26,2011, the entire teachings of these applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Pulmonary delivery of therapeutic agents can offer several advantagesover other modes of delivery. These advantages include rapid onset ofdrug action, the convenience of patient self-administration, thepotential for reduced drug side-effects, ease of delivery, theelimination of needles, and the like. With these advantages, inhalationtherapy is capable of providing a drug delivery system that is easy touse in an inpatient or outpatient setting.

Metered dose inhalers (MDIs) are used to deliver therapeutic agents tothe respiratory tract. MDIs are generally suitable for administeringtherapeutic agents that can be formulated as solid respirable dryparticles in a volatile liquid under pressure. Opening of a valvereleases the suspension at relatively high velocity. The liquid thenvolatilizes, leaving behind a fast-moving aerosol of dry particles thatcontain the therapeutic agent. MDIs are reliable for drug delivery tothe upper and middle airways but are limited because they typicallydeliver only low doses per actuation. However, it is the bronchioles andalveoli that are often the site of manifestation of pulmonary diseasessuch as asthma and respiratory infections.

Liquid aerosol delivery is one of the oldest forms of pulmonary drugdelivery. Typically, liquid aerosols are created by an air jetnebulizer, which releases compressed air from a small orifice at highvelocity, resulting in low pressure at the exit region due to theBernoulli effect. See, e.g., U.S. Pat. No. 5,511,726. The low pressureis used to draw the fluid to be aerosolized out of a second tube. Thisfluid breaks into small droplets as it accelerates in the air stream.Disadvantages of this standard nebulizer design include relatively largeprimary liquid aerosol droplet size often requiring impaction of theprimary droplet onto a baffle to generate secondary splash droplets ofrespirable sizes, lack of liquid aerosol droplet size uniformity,significant recirculation of the bulk drug solution, and low densitiesof small respirable liquid aerosol droplets in the inhaled air.

Ultrasonic nebulizers use flat or concave piezoelectric disks submergedbelow a liquid reservoir to resonate the surface of the liquidreservoir, forming a liquid cone which sheds aerosol particles from itssurface (U.S. 2006/0249144 and U.S. Pat. No. 5,551,416). Since noairflow is required in the aerosolization process, high aerosolconcentrations can be achieved, however the piezoelectric components arerelatively expensive to produce and are inefficient at aerosolizingsuspensions, requiring active drug to be dissolved at low concentrationsin water or saline solutions. Newer liquid aerosol technologies involvegenerating smaller and more uniform liquid respirable dry particles bypassing the liquid to be aerosolized through micron-sized holes. See,e.g., U.S. Pat. No. 6,131,570; U.S. Pat. No. 5,724,957; and U.S. Pat.No. 6,098,620. Disadvantages of this technique include relativelyexpensive piezoelectric and fine mesh components as well as fouling ofthe holes from residual salts and from solid suspensions.

Dry powder inhalation has historically relied on lactose blending toallow for the dosing of particles that are small enough to be inhaled,but aren't dispersible enough on their own. This process is known to beinefficient and to not work for some drugs. For example, the drugloading in the overall dry powder is low due to the presence of thelactose carrier which is typically large and bulky. Several groups havetried to improve on these shortcomings by developing dry powder inhaler(DPI) formulations that are respirable and dispersible and thus do notrequire lactose blending. Dry powder formulations for inhalation therapyare described in U.S. Pat. No. 5,993,805 to Sutton et al.; U.S. Pat. No.6,921,6527 to Platz et al.; WO 0000176 to Robinson et al.; WO 9916419 toTarara et al.; WO 0000215 to Bot et al; U.S. Pat. No. 5,855,913 to Haneset al.; and U.S. Pat. Nos. 6,136,295 and 5,874,064 to Edwards et al.

Broad clinical application of dry powder inhalation delivery has beenlimited by difficulties in generating dry powders of appropriateparticle size, particle density, and dispersibility, in keeping the drypowder stored in a dry state, and in developing a convenient, hand-helddevice that effectively disperses the respirable dry particles to beinhaled in air. In addition, the particle size of dry powders forinhalation delivery is inherently limited by the fact that smallerrespirable dry particles are harder to disperse in air. Dry powderformulations, while offering advantages over cumbersome liquid dosageforms and propellant-driven formulations, are prone to aggregation andlow flowability which considerably diminish dispersibility and theefficiency of dry powder-based inhalation therapies. For example,interparticular Van der Waals interactions and capillary condensationeffects are known to contribute to aggregation of dry particles. Hickey,A. et al., “Factors Influencing the Dispersion of Dry Powders asAerosols”, Pharmaceutical Technology, August, 1994.

The propensity for particles to aggregate or agglomerate increases asparticle size decreases. In order to deaggregate particles of a smallersize, a relatively larger dispersion energy is needed. This can bedescribed as inhaled flowrate dependency since the degree of dispersionof the agglomerated particles is a function of inhaled flowrate. Whatthis means to a clinician and a patient is that the dose the patientreceives varies depending on their inspiratory flowrate.

One example of how the art has dealt with the need for a high dispersionenergy is to require the patient to inhale on a passive dry powderinhaler (DPI) at a high inspiratory flow rate. In Anderson, et al,(European Respiratory Journal, 1997, November; 10(11):2465-73)micronized sodium chloride was delivered to patients to causebroncho-provocation. Patients were required to breathe forcefully on theDPI in order to receive the broncho-provocative dose. Flowrates ofgreater than or equal to 50 LPM on a standard DPI and greater than 28LPM on a high-resistance DPI were required, both produce higherdispersion energies.

Requiring a patient to inspire at a high flowrate is not alwayspossible, or predictable, e.g., due to patient's disease state orphysical condition. Previously, the problem of delivering active agentsto the respiratory tract at a relatively constant dose across variousflowrates was addressed i) by adding large carrier particles (e.g.,typically with an average particle size in excess of 40 μm), such aslactose, ii) by manufacturing particles that are large and porous (e.g.,tap density of less than 0.4 g/cc), or iii) by using active dry powderdevices that apply significant force to disperse the powders. The firstmethod is still subject to significant variability at varyinginspiratory flowrates. The second method requires large volumes ofpowder to delivery a relatively large dose of powder. The third methodrequires an expensive inhaler to be purchased, that may also be subjectto technical failure. Lipp et al. in U.S. Pat. No. 7,807,200 discuss theproduction of dry particles having low tap densities to avoidaggregation, e.g., tap densities of less than about 0.4 g/cc andpreferably less than about 0.1 g/cc.

To overcome interparticle adhesive forces, Batycky et al. in U.S. Pat.No. 7,182,961 teach production of so called “aerodynamically lightrespirable particles,” which have a volume median geometric diameter(VMGD) of greater than 5 microns (μm) as measured using a laserdiffraction instrument such as HELOS (manufactured by Sympatec,Princeton, N.J.) and a tap density of less than 0.4 g/cc. See Batycky etal., column 7, lines 42-65. Another approach to improve dispersibilityof respirable particles of average particle size of less than 10 μm,involves the addition of a water soluble polypeptide or addition ofsuitable excipients (including amino acid excipients such as leucine) inan amount of 50% to 99.9% by weight of the total composition. Eljamal etal., U.S. Pat. No. 6,582,729, column 4, lines 12-19 and column 5, line55 to column 6, line 31. However, this approach reduces the amount ofactive agent that can be delivered using a fixed amount of powder.Therefore, an increased amount of dry powder is required to achieve theintended therapeutic results, for example, multiple inhalations and/orfrequent administration may be required. Still other approaches involvethe use of devices that apply mechanical forces, such as pressure fromcompressed gasses, to the small particles to disrupt interparticularadhesion during or just prior to administration. See, e.g., U.S. Pat.Nos. 7,601,336 to Lewis et al., 6,737,044 to Dickinson et al., 6,546,928to Ashurst et al., or U.S. Pat. Applications 20090208582 to Johnston etal.

A further limitation that is shared by each of the above methods is thatthe aerosols produced typically include substantial quantities of inertcarriers, solvents, emulsifiers, propellants, and other non-drugmaterial. In general, large quantities of non-drug material are requiredfor effective formation of respirable dry particles small enough foralveolar delivery (e.g., less than 5 microns and preferably less than 3microns). However, these amounts of non-drug material also serve toreduce the purity and amount of active drug substance that can bedelivered. Thus, these methods remain substantially incapable ofintroducing large active drug dosages accurately to a patient forsystemic delivery.

Therefore, there remains a need for the formation of small particle sizeaerosols that are highly dispersible. In addition, methods that produceaerosols comprising greater quantities of drug and lesser quantities ofnon-drug material are needed. Finally, a method that allows a patient toadminister a unit dosage rapidly with one or two, small volume breathsis needed.

SUMMARY OF THE INVENTION

The invention relates to respirable dry particles that contain adivalent metal cation salt, such as calcium (Ca²⁺), in an amount of lessthan 3% by weight, and to dry powders that contain the respirableparticles. The invention also relates to respirable dry powders thatcomprise dry particles that contain less than 3% by weight divalentmetal cation, one or more monovalent cations, and an active agent. Thedivalent cation is generally present in the dry powders and dryparticles in the form of one or more salts, which can independently becrystalline, amorphous or a combination of crystalline and amorphous.The dry powders and dry particles can optionally include additionalmonovalent salts (e.g., sodium salts), therapeutically active agents orpharmaceutically acceptable excipients. In one aspect, the respirabledry particles may be small and highly dispersible. In another aspect,the respirable dry particles may be large or small, e.g., a geometricdiameter (VMGD) between 0.5 microns and 30 microns. Optionally, the MMADof the particles may be between 0.5 and 10 microns, more preferablybetween 1 and 5 microns.

In some aspects, the respirable dry powders have a volume mediangeometric diameter (VMGD) of about 10 microns or less and adispersibility ratio (ratio of VMGD measured at dispersion pressure of 1bar to VMGD measured at 4 bar (1/4 bar)) of less than about 2 asmeasured by laser diffraction (RODOS/HELOS system), and contain acalcium salt that provides divalent metal cation in an amount less than3% by weight of the dry powder. The respirable dry powders can furthercomprise a monovalent salt that provides monovalent cation, such as Na⁺,in an amount of about 6% or more by weight of the powders.

The respirable dry powders can have a Fine Particle Fraction (FPF) ofless than 5.6 microns of at least 45% of the total dose, FPF of lessthan 3.4 microns of at least 30% of the total dose, and/or FPF of lessthan 5.0 microns of at least 45% of the total dose. Alternatively or inaddition, the respirable dry powders can have a mass median aerodynamicdiameter (MMAD) of about 5 microns or less. The molecular weight ratioof divalent metal cation to the divalent metal cation salt contained inthe respirable dry particle can be greater than about 0.1 and/or greaterthan about 0.16.

The respirable dry powder compositions can include a pharmaceuticallyacceptable excipient, such as leucine, maltodextrin or mannitol.

The divalent metal cation salt present in the respirable dry powders canbe a beryllium salt, a magnesium salt, a calcium salt, a strontium salt,a barium salt, a radium salt and a ferrous salt. For example, thedivalent metal cation salt can be a calcium salt, such as calciumlactate, calcium sulfate, calcium citrate, calcium chloride or anycombination thereof. The monovalent salt that is optionally present inthe respirable dry particle can be a sodium salt, a lithium salt, apotassium salt or any combination thereof.

In certain aspects, the respirable dry powder contains a divalent metalcation salt and a monovalent salt, and contains an amorphous divalentmetal cation phase and a crystalline monovalent salt phase. The glasstransition temperature of the amorphous phase can be least about 80° C.These respirable dry particles can optionally contain an excipient, suchas leucine, maltodextrin and mannitol, which can be amorphous,crystalline or a mixture of forms. The respirable dry particle can havea heat of solution between about −10 kcal/mol and 10 kcal/mol.

Preferably, the divalent metal cation salt is a calcium, and themonovalent salt is a sodium salt. The calcium salt can be calciumcitrate, calcium lactate, calcium sulfate, calcium chloride or anycombination thereof, and the sodium salt can be sodium chloride.Alternatively, the calcium salt can be calcium carbonate. In anotheraspect, the divalent metal cation is a magnesium salt. In this aspect,the presence is a monovalent salt is optional.

In other aspects, the respirable dry powder contains a divalent metalsalt that provides a cation in an amount less than 3% by weight of thedry powder, the respirable dry powder has a Hausner Ratio of greaterthan 1.5 and a dispersibility ratio of 1/4 bar or 0.5/4 bar of 2 orless.

The invention also relates to methods for treating a respiratorydisease, such as asthma, airway hyperresponsiveness, seasonal allergicallergy, bronchiectasis, chronic bronchitis, emphysema, chronicobstructive pulmonary disease, cystic fibrosis and the like, comprisingadministering to the respiratory tract of a subject in need thereof aneffective amount of the respirable dry particles or dry powder. Theinvention also relates to methods for the treatment or prevention ofacute exacerbations of chronic pulmonary diseases, such as asthma,airway hyperresponsiveness, seasonal allergic allergy, bronchiectasis,chronic bronchitis, emphysema, chronic obstructive pulmonary disease,cystic fibrosis and the like, comprising administering to therespiratory tract of a subject in need thereof an effective amount ofthe respirable dry particles or dry powder.

The invention also relates to methods for treating, preventing and/orreducing contagion of an infectious disease of the respiratory tract,comprising administering to the respiratory tract of a subject in needthereof an effective amount of the respirable dry particles or drypowder.

The invention also relates to a respirable dry powder or dry particle,as described herein, for use in therapy (e.g., treatment, prophylaxis,or diagnosis). The invention also relates to the use of a respirable dryparticle or dry powder, as described herein, for use in treatment,prevention or reducing contagion as described herein, and in themanufacture of a medicament for the treatment, prophylaxis or diagnosisof a respiratory disease and/or infection as described herein.

The invention relates to respirable dry particles that contain one ormore divalent metal cations, such as calcium or magnesium, in an amountof less than 3% by weight of respirable dry particle, and to dry powdersthat contain these respirable particles. The dry particles and the drypowders can further contain one or more pharmaceutically activeagent(s), e.g., therapeutic and/or prophylactic agents. For example, thedry particles can contain one or more active agent(s) in aco-formulation. The dry particles and active agent(s) can beco-formulated, e.g., by spray drying, freeze-drying, super criticalfluids, etc. In another example, the dry particles are not co-formulatedand can be used as carrier particles to deliver one or more activeagent(s). The carrier particles may be blended together with the one ormore active agent(s) to produce a dry powder. The active agent(s) may bein micronized form. Alternatively or in addition, the dry particles maybe co-formulated with an active agent(s) (e.g., comprising a first,second, etc. active agent) and subsequently used as carrier particlesfor additional active agent(s) (e.g., a second, third, fourth, etc.active agent). The co-formulated dry particles may be blended with theone or more additional active agent(s). The resulting dry powdercontains both the co-formulated dry particles and the blended activeagent. The one or more additional active agent(s) can be the same activeagent(s) that are co-formulated in the dry particle, different activeagents, or a combination thereof.

Suitable active agents include, but are not limited to, mucoactive ormucolytic agents, surfactants, antibiotics, antivirals, antihistamines,cough suppressants, bronchodilators, anti-inflammatory agents, steroids,vaccines, adjuvants, expectorants, macromolecules, or therapeutics thatare helpful for chronic maintenance of cystic fibrosis (CF). Preferredactive agents include, but are not limited to, LABAs (e.g., formoterol,salmeterol), short-acting beta agonists (e.g., albuterol),corticosteroids (e.g., fluticasone), LAMAs (e.g., tiotropium),antibiotics (e.g., levofloxacin, tobramycin), antibodies (e.g.,therapeutic antibodies), hormones (e.g., insulin), chemokines (e.g.,cytokines), growth factors, and combinations thereof. When the drypowders are intended for treatment of CF, preferred additional activeagents are short-acting beta agonists (e.g., albuterol), antibiotics(e.g., levofloxacin), recombinant human deoxyribonuclease I (e.g.,dornase alfa, also known as DNase), sodium channel blockers (e.g.,amiloride), and combinations thereof.

The respirable dry particles of the invention are small and dispersible,and can be used to administer active agents to the lungs, including thedeep lung, for local action in the lung or for absorption through thelung and systemic action. Optionally, the MMAD of the dry powder may bebetween 0.5 and 10 microns, more preferably between 1 and 5 microns,between 1 and 3 microns or between 3 and 5 microns.

In one aspect, the respirable dry powders and dry particles of theinvention may be active agent dense, small and dispersible. For example,the dry particles can contain a high percentage of one or morepharmaceutically active agents. For example, as described herein, theactive agent may comprise 5% or more, 10% or more, 20% or more, 30% ormore, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more,90% or more, 95% or more, or 97% or more by weight of the dry particle.

In another aspect, the respirable dry particles are mass dense (e.g.,have a tap density or envelope mass density of greater than about 0.4g/cc, or at least about 0.45 g/cc or greater, about 0.5 g/cc or greater,about 0.55 g/cc or greater, about 0.6 g/cc or greater, about 0.7 g/cc orgreater or about 0.8 g/cc or greater), small, and dispersible.

The respirable dry particles are generally small, e.g., they possess ageometric diameter (VMGD) between 0.5 microns and 10 microns, or between1 micron and 7 microns. Optionally, the MMAD of the dry powder may bebetween 0.5 and 10 microns, more preferably between 1 and 5 microns. Theparticles optionally have a tap density or envelope mass density greaterthan 0.4 g/cc, between 0.45 g/cc and 1.2 g/cc, or between 0.55 g/cc and1.0 g/cc.

The respirable dry particles may also be large, e.g., they posses a(VMGD) between 10 microns and 30 microns, or between 10 microns and 20microns. Optionally, the MMAD of the dry powder may be between 0.5 and10 microns, more preferably between 1 and 5 microns. The particlesoptionally have a tap density or envelope mass density between 0.01 g/ccand 0.4 g/cc, or between 0.05 g/cc and 0.25 g/cc. They are alsogenerarally dispersible.

Respirable dry powders that contain small particles and that aredispersible in air, and preferably dense (e.g., dense in active agent)are a departure from the conventional wisdom. It is well known that thepropensity for particles to aggregate or agglomerate increases asparticle size decreases. See, e.g., Hickey, A. et al., “FactorsInfluencing the Dispersion of Dry Powders as Aerosols”, PharmaceuticalTechnology, August, 1994.

As described herein, the invention provides respirable dry powders thatcontain respirable particles that are small and dispersible in airwithout additional energy sources beyond the subject's inhalation. Thus,the respirable dry powders and respirable dry particles can be usedtherapeutically, without including large amounts of non-activecomponents (e.g., excipients such as lactose carrier particles) in theparticles or powders, or by using devices that apply mechanical forcesto disrupt aggregated or agglomerated particles during or just prior toadministration. Rather, devices, such as a passive dry powder inhaler,may be used to deliver the dry powder or dry particles described herein.In some embodiments, the respirable dry powders and respirable dryparticles do not include any excipient (e.g., leucine) in the particlesor powders.

The respirable dry powders and respirable particles of the invention canbe dense in active agent(s). Suitable active agents include, but are notlimited to, mucoactive or mucolytic agents, surfactants, antibiotics,antivirals, antihistamines, cough suppressants, bronchodilators,anti-inflammatory agents, steroids, vaccines, adjuvants, expectorants,macromolecules, or therapeutics that are helpful for chronic maintenanceof cystic fibrosis (CF). Preferred active agents include, but are notlimited to, LABAs (e.g., formoterol, salmeterol), short-acting betaagonists (e.g., albuterol), corticosteroids (e.g., fluticasone), LAMAs(e.g., tiotropium), antibiotics (e.g., levofloxacin, tobramycin),antibodies (e.g., therapeutic antibodies), hormones (e.g., insulin),chemokines, cytokines, growth factors, and combinations thereof. Whenthe dry powders are intended for treatment of CF, preferred additionalactive agents are short-acting beta agonists (e.g., albuterol),antibiotics (e.g., levofloxacin), recombinant human deoxyribonuclease I(e.g., dornase alfa, also known as DNase), sodium channel blockers(e.g., amiloride), and combinations thereof.

Accordingly, for respirable dry powders and respirable particles thatare dense in active agent(s), a smaller amount of powder will need to beadministered to a subject (e.g., a patient) in order to deliver thedesired dose of active agent, in comparison to conventional dry powders,such as powders that contain lactose carrier particles. For example, thedesired dose of active agent may be delivered with one or twoinhalations from a capsule-type or blister-type inhaler using the activeagent dense dry powders or particles described herein, whereas three,four or more inhalations may be necessary to administer to a subject anactive agent that is present in conventional large porous particles.Hence, respirable dry particles and dry powders that are small,dispersible and dense (e.g., dense in active agent, and/or mass dense)provide significant advantages over the powders commonly used in theart.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E are graphs illustrating the aerodynamic particle sizedistribution of exemplary dry powders of the invention as measured by aneight stage Anderson cascade impactor (ACI). The graphs indicate thatall five dry powders were of a respirable size.

FIG. 2 is a graph illustrating the efficacy of a divalent cation-baseddry powder formulation of tiotroprium bromide (TioB) in reducing airwayhyperreactivity in an ovalbumin (OVA) mouse model of allergic asthma.The graph indicates that the spray dried drug (TioB) remained effectivein treating airway hyperreactivity.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to respirable dry particles that contain one ormore divalent metal cations, such as calcium, in an amount of less than3% by weight, and to dry powders that contain the respirable particles.The dry particles can further contain an active agent (such asmucoactive or mucolytic agents, surfactants, antibiotics, antivirals,antihistamines, cough suppressants bronchodilators anti-inflammatoryagents, steroids, vaccines, adjuvants, expectorants, macromolecules, ortherapeutics that are helpful for chronic maintenance of cystic fibrosis(CF)), or can be used as carrier particles to deliver an active agent.The respirable dry particles of the invention are small and dispersible,and can be used to administer active agents to the lungs, including thedeep lung, for local action in the lung or for absorbtion through thelung and systemic action.

In one aspect, the respirable dry powders and dry particles of theinvention may be active agent dense, small and dispersible. Optionally,the MMAD of the dry powder may be between 0.5 and 10 microns, morepreferably between 1 and 5 microns.

Respirable dry powders that contain small particles and that aredispersible in air, and preferably dense (e.g., dense in active agent)are a departure from the conventional wisdom. It is well known that thepropensity for particles to aggregate or agglomerate increases asparticle size decreases. See, e.g., Hickey, A. et al., “FactorsInfluencing the Dispersion of Dry Powders as Aerosols”, PharmaceuticalTechnology, August, 1994.

As described herein, the invention provides respirable dry powders thatcontain respirable particles that are small and dispersible in airwithout additional energy sources beyond the subject's inhalation. Thus,the respirable dry powders and respirable dry particles can be usedtherapeutically, without including large amounts of non-activecomponents (e.g., excipients such as lactose carrier particles) in theparticles or powders, or by using devices that apply mechanical forcesto disrupt aggregated or agglomerated particles during or just prior toadministration. In some embodiments, the respirable dry powders andrespirable dry particles do not include any excipient (e.g., leucine) inthe particles or powders.

The respirable dry powders and respirable particles of the invention canbe dense in active agent(s), e.g., mucoactive or mucolytic agents,surfactants, antibiotics, antivirals, antihistamines, coughsuppressants, bronchodilators, anti-inflammatory agents, steroids,vaccines, adjuvants, expectorants, macromolecules, or therapeutics thatare helpful for chronic maintenance of CF.

For example, as described herein, when an excipient is included in therespirable dry powder or particles, the excipient may comprise about 50%or less by weight, or about 40% or less by weight, or about 30% or lessby weight, or about 20% or less by weight, about 12% or less by weight,about 10% or less by weight, about 8% or less by weight). Thus, in oneaspect, the respirable particles are not only small and highlydispersible, but can contain a large amount of active ingredient.Accordingly, a smaller amount of powder will need to be administered inorder to deliver the desired dose of active agent, in comparison toconventional dry powders, such as powders that contain lactose carrierparticles. For example, the desired dose of active agent may bedelivered with one or two inhalations from a capsule-type orblister-type inhaler.

DEFINITIONS

The term “dry powder” as used herein refers to a composition thatcontains finely dispersed respirable dry particles that are capable ofbeing dispersed in an inhalation device and subsequently inhaled by asubject. Such a dry powder may contain up to about 25%, up to about 20%,or up to about 15% water or other solvent, or be substantially free ofwater or other solvent, or be anhydrous.

The term “dry particles” as used herein refers to respirable particlesthat may contain up to about 25%, up to about 20%, or up to about 15%water or other solvent, or be substantially free of water or othersolvent, or be anhydrous.

The term “respirable” as used herein refers to dry particles or drypowders that are suitable for delivery to the respiratory tract (e.g.,pulmonary delivery) in a subject by inhalation. Respirable dry powdersor dry particles have a mass median aerodynamic diameter (MMAD) of lessthan about 10 microns, preferably about 5 microns or less.

The term “small” as used herein to describe respirable dry particlesrefers to particles that have a volume median geometric diameter (VMGD)of about 10 microns or less, preferably about 5 microns or less.

As used herein, the terms “administration” or “administering” ofrespirable dry particles refers to introducing respirable dry particlesto the respiratory tract of a subject.

As used herein, the term “respiratory tract” includes the upperrespiratory tract (e.g., nasal passages, nasal cavity, throat, andpharynx), respiratory airways (e.g., larynx, tranchea, bronchi, andbronchioles) and lungs (e.g., respiratory bronchioles, alveolar ducts,alveolar sacs, and alveoli).

The term “dispersible” is a term of art that describes thecharacteristic of a dry powder or dry particles to be dispelled into arespirable aerosol. Dispersibility of a dry powder or dry particles isexpressed herein as the quotient of the volume median geometric diameter(VMGD) measured at a dispersion (i.e., regulator) pressure of 1 bardivided by the VMGD measured at a dispersion (i.e., regulator) pressureof 4 bar, or VMGD at 0.5 bar divided by the VMGD at 4 bar as measured byHELOS/RODOS laser diffraction system. These quotients are referred toherein as “1 bar/4 bar,” and “0.5 bar/4 bar,” respectively, anddispersibility correlates with a low quotient. For example, 1 bar/4 barrefers to the VMGD of respirable dry particles or powders emitted fromthe orifice of a RODOS dry powder disperser (or equivalent technique) atabout 1 bar, as measured by a HELOS or other laser diffraction system,divided the VMGD of the same respirable dry particles or powdersmeasured at 4 bar by HELOS/RODOS. Thus, a highly dispersible dry powderor dry particles will have a 1 bar/4 bar or 0.5 bar/4 bar ratio that isclose to 1.0. Highly dispersible powders have a low tendency toagglomerate, aggregate or clump together and/or, if agglomerated,aggregated or clumped together, are easily dispersed or de-agglomeratedas they emit from an inhaler and are breathed in by the subject.Dispersibility can also be assessed by measuring the size emitted froman inhaler as a function of flowrate. As the flow rate through theinhaler decreases, the amount of energy in the airflow available to betransferred to the powder to disperse it decreases. A highly dispersiblepowder will have its size distribution as characterized aerodynamicallyby its mass median aerodynamic diameter (MMAD) or geometrically by itsVMGD, not substantially decrease over a range of flow rates typical ofinhalation by humans, such as from about 15 to about 60 LPM (L/min orliters per minute).

The terms “FPF (<5.6),” “FPF (<5.6 microns),” and “fine particlefraction of less than 5.6 microns” as used herein, refer to the fractionof a sample of dry particles that have an aerodynamic diameter of lessthan 5.6 microns. For example, FPF (<5.6) can be determined by dividingthe mass of respirable dry particles deposited on the stage one and onthe collection filter of a two-stage collapsed Andersen Cascade Impactor(ACI) by the mass of respirable dry particles weighed into a capsule fordelivery to the instrument. This parameter may also be identified as“FPF_TD(<5.6),” where TD means total dose. A similar measurement can beconducted using an eight-stage ACI. The eight-stage ACI cutoffs aredifferent at the standard 60 L/min flowrate, but the FPF_TD(<5.6) can beextrapolated from the eight-stage complete data set. The eight-stage ACIresult can also be calculated by the USP (United States Pharmacopeiaconvention) method of using the dose collected in the ACI instead ofwhat was in the capsule to determine FPF.

The terms “FPF (<3.4),” “FPF (<3.4 microns),” and “fine particlefraction of less than 3.4 microns” as used herein, refer to the fractionof a mass of respirable dry particles that have an aerodynamic diameterof less than 3.4 microns. For example, FPF (<3.4) can be determined bydividing the mass of respirable dry particles deposited on thecollection filter of a two-stage collapsed ACI by the total mass ofrespirable dry particles weighed into a capsule for delivery to theinstrument. This parameter may also be identified as “FPF_TD(<3.4),”where TD means total dose. A similar measurement can be conducted usingan eight-stage ACI. The eight-stage ACI result can also be calculated bythe USP method of using the dose collected in the ACI instead of whatwas in the capsule to determine FPF.

The terms “FPF (<5.0),” “FPF (<5.0 microns),” and “fine particlefraction of less than 5.0 microns” as used herein, refer to the fractionof a mass of respirable dry particles that have an aerodynamic diameterof less than 5.0 microns. For example, FPF (<5.0) can be determined byusing an eight-stage ACI at the standard 60 L/min flowrate byextrapolating from the eight-stage complete data set. This parameter mayalso be identified as “FPF_TD(<5.0),” where TD means total dose. Whenused in conjunction with a geometric size distribution such as thosegiven by a Malvern Spraytec, Malvern Mastersizer or Sympatec HELOSparticle sizer, “FPF (<5.0)” refers to the fraction of a mass ofrespirable dry particles that have a geometric diameter of less than 5.0micrometers.

The terms “FPD(<4.4)”, “FPD<4.4 μm”, “FPD(<4.4 microns)” and “fineparticle dose of less than 4.4 microns” as used herein, refer to themass of respirable dry powder particles that have an aerodynamicdiameter of less than 4.4 micrometers. For example, FPD<4.4 μm can bedetermined by using an eight-stage ACI at the standard 60 L/min flowrateand summing the mass deposited on the filter, and stages 6, 5, 4, 3, and2 for a single dose of powder actuated into the ACI.

As used herein, the term “emitted dose” or “ED” refers to an indicationof the delivery of a drug formulation from a suitable inhaler deviceafter a firing or dispersion event. More specifically, for dry powderformulations, the ED is a measure of the percentage of powder that isdrawn out of a unit dose package and that exits the mouthpiece of aninhaler device. The ED is defined as the ratio of the dose delivered byan inhaler device to the nominal dose (i.e., the mass of powder per unitdose placed into a suitable inhaler device prior to firing). The ED isan experimentally-measured parameter, and can be determined using themethod of USP Section 601 Aerosols, Metered-Dose Inhalers and Dry PowderInhalers, Delivered-Dose Uniformity, Sampling the Delivered Dose fromDry Powder Inhalers, United States Pharmacopia convention, Rockville,Md., 13^(th) Revision, 222-225, 2007. This method utilizes an in vitrodevice set up to mimic patient dosing.

The term “capsule emitted powder mass” or “CEPM” as used herein, refersto the amount of dry powder formulation emitted from a capsule or doseunit container during an inhalation maneuver. CEPM is measuredgravimetrically, typically by weighing a capsule before and after theinhalation maneuver to determine the mass of powder formulation removed.CEPM can be expressed either as the mass of powder removed, inmilligrams, or as a percentage of the initial filled powder mass in thecapsule prior to the inhalation maneuver.

The term “effective amount,” as used herein, refers to the amount ofactive agent needed to achieve the desired therapeutic or prophylacticeffect, such as an amount that is sufficient to reduce pathogen (e.g.,bacteria, virus) uptake or pathogen burden, reduce symptoms (e.g.,fever, coughing, sneezing, nasal discharge, diarrhea and the like),reduce occurrence of infection, reduce viral replication, or improve orprevent deterioration of respiratory function (e.g., improve forcedexpiratory volume in 1 second FEV1 and/or forced expiratory volume in 1second FEV1 as a proportion of forced vital capacity FEV1/FVC), orproduce an effective serum concentration of a pharmaceutically activeagent. The actual effective amount for a particular use can varyaccording to the particular dry powder or dry particle, the mode ofadministration, and the age, weight, general health of the subject, andseverity of the symptoms or condition being treated. Suitable amounts ofdry powders and dry particles to be administered, and dosage schedulesfor a particular patient can be determined by a clinician of ordinaryskill based on these and other considerations.

In certain preferred embodiments, the effective amount is sufficient toincrease surface and/or bulk viscoelasticy of the respiratory tractmucus (e.g., airway lining fluid), increase gelation of the respiratorytract mucus (e.g., at the surface and/or bulk gelation), increasesurface tension of the respiratory tract mucus, increasing elasticity ofthe respiratory tract mucus (e.g., surface elasticity and/or bulkelasticity), increase surface viscosity of the respiratory tract mucus(e.g., surface viscosity and/or bulk viscosity), reduce the amount ofexhaled particles, and/or stimulate innate immunity of airwayepithelium.

The term “pharmaceutically acceptable excipient” as used herein meansthat the excipient can be taken into the lungs with no significantadverse toxicological effects on the lungs. Such excipients aregenerally regarded as safe (GRAS) by the U.S. Food and DrugAdministration.

All references to salts herein include anhydrous forms and all hydratedforms of the salt.

All weight percentages are given on a dry basis.

Dry Powders and Dry Particles

The invention relates to respirable dry powders and dry particles thatcontain one or more divalent metal cations in an amount of less than 3%by weight of dry particle, and optionally, one or more monovalent metalcation salts.

The respirable dry powder and dry particles of the invention contain alow percentage of divalent metal cation (e.g., calcium, magnesium). Thedry particles contain less than 3% divalent metal cation by weight. Forexample, the dry particles can contain between about 0.1% and 2.9%divalent metal cation by weight.

In a preferred aspect of the invention, the respirable dry powders orrespirable dry particles are suitable for administering an active agentto a patient. The dry particles can i) contain one or more activeagent(s) in a co-formulation, ii) the dry particles can be used ascarrier particles to deliver one or more active agent(s), or iii) thedry particles can be co-formulated with one or more active agent(s)(e.g., first, second, etc. active agent) and used as carrier particlesto deliver one or more additional active agent(s) (e.g., second, third,fourth, etc. active agent). Active agents include, but are not limitedto, mucoactive or mucolytic agents, surfactants, antibiotics,antivirals, antihistamines, cough suppressants, bronchodilators,anti-inflammatory agents, steroids, vaccines, adjuvants, expectorants,macromolecules, or therapeutics that are helpful for chronic maintenanceof cystic fibrosis (CF). Preferred active agents include, but are notlimited to, LABAs (e.g., formoterol, salmeterol), short-acting betaagonists (e.g., albuterol), corticosteroids (e.g., fluticasone), LAMAs(e.g., tiotropium), antibiotics (e.g., levofloxacin, tobramycin),antibodies (e.g., therapeutic antibodies), hormones (e.g., insulin),chemokines, cytokines, growth factors, and combinations thereof. Whenthe dry powders are intended for treatment of CF, preferred additionalactive agents are short-acting beta agonists (e.g., albuterol),antibiotics (e.g., levofloxacin), recombinant human deoxyribonuclease I(e.g., dornase alfa, also known as DNase), sodium channel blockers(e.g., amiloride), and combinations thereof.

The dry particles and active agent(s) can be co-formulated.Co-formulating an active agent into a solution or suspension thatcontains the divalent cation and, optionally, other components, and thenprocessing the solution or suspension into dry particles e.g., by spraydrying, freeze drying, etc., is a preferred aspect of the invention.

Alternatively, the divalent cation salt, optionally with one or moremonovalent salt(s) and/or excipients, can be manufactured (e.g., viaspray drying) to make dry particles. These dry particles cansubsequently be combined with an active agent, e.g., by blending the dryparticle with one or more (micronized) therapeutic agents. The dryparticles may act as carrier particles in the dry powder when blendedtogether with the (micronized) active agent.

Alternatively or in addition, co-formulated dry particles (containingactive agent(s)) can further be blended with additional (micronized)active agents resulting in a dry powder containing co-formulated dryparticles (containing active agent(s)) and additional (micronized)active agent(s) in a blend. The co-formulated dry particles may act ascarrier particles for the (micronized) active agent in the dry powder.

Chemical Composition

In one aspect, the respirable dry particles of the invention contain oneor more divalent metal cation salts (e.g., a calcium salt and/or amagnesium salt), and optionally one or more monovalent metal cationsalts (e.g., a sodium salt and/or a potassium salt) and/or an excipient,but do not contain an active agent. These types of respirable dryparticles can be blended with a (micronized) active agent to produce adry powder of the invention. This type of respirable dry particle can beused as carrier particles to deliver an active agent to the respiratorytract (e.g., lungs) for local or systemic delivery.

In another, preferred aspect, the respirable dry particles of theinvention contain one or more divalent metal cation salts (e.g., acalcium salt and/or a magnesium salt), and optionally one or moremonovalent metal cation salts (e.g., a sodium salt and/or a potassiumsalt) and/or an excipient, and further contain an active agent in aco-formulation. These types of respirable dry particles can be prepared,for example, by spray drying a feed stock that contains the divalentmetal cation salt, the active agent and optionally a monovalent metalcation salt and/or an excipient, as described herein. The co-formulateddry particles can be used to deliver a pharmaceutically active agent tothe respiratory tract (e.g., lungs) for local or systemic delivery.

Alternatively or in addition, the co-formulated particle containing afirst active agent can then be used as a carrier particle for a secondactive agent in the dry powder, e.g., when the co-formulated dryparticle is blended with the second active agent (e.g., an agent inmicronized form). This type of co-formulated respirable dry particle canbe used also as a carrier particle to deliver an active agent to therespiratory tract (e.g., lungs) for local or systemic delivery.

It is generally preferred that the one or more divalent metal cationsthat are present in the dry particle in a total amount of less than 3%by weight of dry particle do not produce a prophylactic or therapeuticeffect in the absence of an active agent when the dry particles areadministered to a subject. However, without wishing to be bound by anyparticular theory, it is believed that in some circumstances such dryparticles may be administered to a subject in a quantity (dose)sufficient for the one or more divalent metal cations that are presentin the dry particle to provide a prophylactic and/or therapeutic effect.It is also believed that the divalent metal cation present in the dryparticle may beneficially affect the activity of the active agent, forexample, the one or more divalent metal cations may potentiate theprophylactic or therapeutic activity of the active agent.

Preferably, the prophylactic and/or therapeutic effect is a biologicalactivity selected from anti-bacterial activity, anti-viral activity,anti-inflammatory activity, mucociliary clearance, and combinationsthereof. Whether a metal cation, on its own, has such a prophylacticand/or therapeutic effect can be evaluated using the in vivo modelsdescribed herein and those known in the art.

Suitable divalent metal cations include beryllium (Be²⁺), magnesium,(Mg²⁺), calcium (Ca²⁺) strontium (Sr²⁺), barium (Ba²⁺), radium (Ra²⁺),zinc (Zn²⁺) or iron (ferrous ion, Fe²⁺). The divalent metal cation(e.g., calcium) is generally present in the dry powders and dryparticles in the form of a salt, which can be crystalline or amorphous.In one aspect, the divalent metal cation can be present in the drypowders and dry particles in the form of a salt, including hydrates orsolvates thereof, which can be crystalline or amorphous. The dry powdersand dry particles can optionally include additional salts (e.g.,monovalent salts, such as sodium salts, potassium salts, and lithiumsalts.), therapeutically active agents or pharmaceutically acceptableexcipients.

In some aspects, the respirable dry powder and dry particles contain oneor more salts of a group IIA element (i.e., one or more beryllium salts,magnesium salts, calcium salts, barium salts, radium salts or anycombination of the foregoing). In more particular aspects, therespirable dry powder and dry particles contain one or more calciumsalts, magnesium salts or any combination of the foregoing. Inparticular embodiments, the respirable dry powder and dry particlescontain one or more calcium salts. In other particular embodiments,respirable dry powder and dry particles contain one or more magnesiumsalts.

Preferred divalent metal salts (e.g., calcium salts) have one,preferably two or more of the following characteristics: (i) can beprocessed into a respirable dry particle, (ii) possess sufficientphysicochemical stability in dry powder form to facilitate theproduction of a powder that is dispersible and physically stable over arange of conditions, including upon exposure to elevated humidity, (iii)undergo rapid dissolution upon deposition in the lungs, for example,half of the mass of the cation of the divalent metal can dissolved inless than 30 minutes, less than 15 minutes, less than 5 minutes, lessthan 2 minutes, less than 1 minute, or less than 30 seconds, and (iv) donot possess properties that can result in poor tolerability or adverseevents, such as a significant exothermic or endothermic heat of solution(ΔH), for example, a ΔH lower than of about −10 kcal/mol or greater thanabout 10 kcal/mol. Rather, a preferred ΔH is between about −9 kcal/moland about 9 kcal/mol, between about −8 kcal/mol and about 8 kcal/mol,between about −7 kcal/mol and about 7 kcal/mol, between about −6kcal/mol and about 6 kcal/mol, between about −5 kcal/mol and about 5kcal/mol, between about −4 kcal/mol and about 4 kcal/mol, between about−3 kcal/mol and about 3 kcal/mol, between about −2 kcal/mol and about 2kcal/mol, between about −1 kcal/mol and about 1 kcal/mol, or about 0kcal/mol.

Suitable beryllium salts include, for example, beryllium phosphate,beryllium acetate, beryllium tartrate, beryllium citrate, berylliumgluconate, beryllium maleate, beryllium succinate, sodium berylliummalate, beryllium alpha brom camphor sulfonate, berylliumacetylacetonate, beryllium formate or any combination thereof.

Suitable magnesium salts include, for example, magnesium fluoride,magnesium chloride, magnesium bromide, magnesium iodide, magnesiumphosphate, magnesium sulfate, magnesium sulfite, magnesium carbonate,magnesium oxide, magnesium nitrate, magnesium borate, magnesium acetate,magnesium citrate, magnesium gluconate, magnesium maleate, magnesiumsuccinate, magnesium malate, magnesium taurate, magnesium orotate,magnesium glycinate, magnesium naphthenate, magnesium acetylacetonate,magnesium formate, magnesium hydroxide, magnesium stearate, magnesiumhexafluorsilicate, magnesium salicylate or any combination thereof.

Suitable calcium salts include, for example, calcium chloride, calciumsulfate, calcium lactate, calcium citrate, calcium carbonate, calciumacetate, calcium phosphate, calcium alginate, calcium stearate, calciumsorbate, calcium gluconate and the like.

Suitable strontium salts include, for example, strontium chloride,strontium phosphate, strontium sulfate, strontium carbonate, strontiumoxide, strontium nitrate, strontium acetate, strontium tartrate,strontium citrate, strontium gluconate, strontium maleate, strontiumsuccinate, strontium malate, strontium aspartate in either L and/orD-form, strontium fumarate, strontium glutamate in either L- and/orD-form, strontium glutarate, strontium lactate, strontium L-threonate,strontium malonate, strontium ranelate (organic metal chelate),strontium ascorbate, strontium butyrate, strontium clodronate, strontiumibandronate, strontium salicylate, strontium acetyl salicylate or anycombination thereof.

Suitable barium salts include, for example, barium hydroxide, bariumfluoride, barium chloride, barium bromide, barium iodide, bariumsulfate, barium sulfide (S), barium carbonate, barium peroxide, bariumoxide, barium nitrate, barium acetate, barium tartrate, barium citrate,barium gluconate, barium maleate, barium succinate, barium malate,barium glutamate, barium oxalate, barium malonate, barium naphthenate,barium acetylacetonate, barium formate, barium benzoate, bariump-t-butylbenzoate, barium adipate, barium pimelate, barium suberate,barium azelate, barium sebacate, barium phthalate, barium isophthalate,barium terephthalate, barium anthranilate, barium mandelate, bariumsalicylate, barium titanate or any combination thereof.

Suitable radium salts included, for example, radium fluoride, radiumchloride, radium bromide, radium iodide, radium oxide, radium nitride orany combination thereof.

Suitable iron (ferrous) salts include, for example, ferrous sulfate,ferrous oxides, ferrous acetate, ferrous citrate, ferrous ammoniumcitrate, ferrous ferrous gluconate, ferrous oxalate, ferrous fumarate,ferrous maleate, ferrous malate, ferrous lactate, ferrous ascorbate,ferrous erythrobate, ferrous glycerate, ferrous pyruvate or anycombination thereof.

As described herein, the respirable dry particles of the inventioncontain one or more divalent metal cations (e.g., calcium (Ca²⁺) and/ormagnesium (Mg²⁺)) which are generally present in the form of a salt inan amount of less than 3% by weight of dry particle. Suitable calciumsalts that can be present in the respirable dry particles of theinvention include, for example, calcium chloride, calcium sulfate,calcium lactate, calcium citrate, calcium carbonate, calcium acetate,calcium phosphate, calcium alginite, calcium stearate, calcium sorbate,calcium gluconate and the like. In certain aspects, the dry powder ordry particles of the invention do not contain calcium phosphate, calciumcarbonate, calcium alginate, calcium sterate or calcium gluconate. Inanother preferred aspect, the dry powder or dry particles of theinvention include calcium citrate, calcium lactate, calcium chloride,calcium sulfate, or any combination of these salts. In another preferredaspect, the dry powder or dry particles include calcium citrate, calciumlactate, or any combination of these salts. In another preferred aspect,the dry powder or dry particles of the invention include calciumlactate, calcium sulfate, calcium carbonate, or any combination of thesesalts. In another aspect, the dry powder or dry particles of theinvention do not contain calcium chloride or calcium phosphate. Ifdesired, the respirable dry particles of the invention contain adivalent metal cation salt (e.g., a calcium salt) and further containone or more additional salts, such as one or more non-toxic salts of theelements sodium, potassium, magnesium, calcium, aluminum, silicon,scandium, titanium, vanadium, chromium, cobalt, nickel, copper,manganese, zinc, tin, silver and the like. If desired, the dry particlescontain at least one calcium salt and at least one monovalent cationsalt (e.g., a sodium salt).

Suitable sodium salts that can be present in the respirable dryparticles of the invention include, for example, sodium chloride, sodiumcitrate, sodium sulfate, sodium lactate, sodium acetate, sodiumbicarbonate, sodium carbonate, sodium stearate, sodium ascorbate, sodiumbenzoate, sodium biphosphate, sodium phosphate, sodium bisulfite, sodiumborate, sodium gluconate, sodium metasilicate and the like. In apreferred aspect, the dry powders and dry particles include sodiumchloride, sodium citrate, sodium lactate, sodium sulfate, or anycombination of these salts.

Suitable lithium salts include, for example, lithium chloride, lithiumbromide, lithium carbonate, lithium nitrate, lithium sulfate, lithiumacetate, lithium lactate, lithium citrate, lithium aspartate, lithiumgluconate, lithium malate, lithium ascorbate, lithium orotate, lithiumsuccinate or and combination thereof.

Suitable potassium salts include, for example, potassium chloride,potassium bromide, potassium iodide, potassium bicarbonate, potassiumnitrite, potassium persulfate, potassium sulfite, potassium bisulfite,potassium phosphate, potassium acetate, potassium citrate, potassiumglutamate, dipotassium guanylate, potassium gluconate, potassium malate,potassium ascorbate, potassium sorbate, potassium succinate, potassiumsodium tartrate and any combination thereof.

Some respirable dry particles contain at least one calcium salt selectedfrom the group consisting of calcium lactate, calcium citrate, calciumsulfate, and calcium chloride, and also contain sodium chloride. Otherrespirable dry particles contain at least one calcium salt selected fromthe group consisting of calcium lactate, calcium citrate and calciumsulfate, and also contain a sodium salt, e.g., sodium chloride. Furtherrespirable dry particles or dry powders contain calcium carbonate. Incertain embodiments, calcium carbonate can be blended with othercomponents into a powder, or can be spray dried as a suspension withother components.

Calcium citrate, calcium sulfate and calcium lactate possess sufficientaqueous solubility to allow for their processing into respirable drypowders via spray-drying and to facilitate their dissolution upondeposition in the lungs, yet possess a low enough hygroscopicity toallow for the production of dry powders with high calcium salt loadsthat are relatively physically stable upon exposure to normal andelevated humidity. Calcium citrate, calcium sulfate and calcium lactatealso have a significantly lower heat of solution than calcium chloride,which is beneficial for administration to the respiratory tract, andcitrate, sulfate and lactate ions are safe and acceptable for inclusionin pharmaceutical compositions.

Accordingly, in addition to any combination of the features andproperties described herein, the respirable dry particles of theinvention can contain a total salt content (e.g., of monovalent anddivalent cation salts) of at least about 51% by weight of the respirabledry particles. For example, the respirable dry particles of theinvention can include one or more of the salts in a total amount of atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, or at leastabout 95% by weight of the respirable dry particles, provided that thedivalent metal cation is present at less than 3% by weight of respirabledry particle.

In another embodiment, the respirable dry particles of the invention cancontain a total salt content (e.g., of monovalent and/or divalent cationsalts) of less than about 51% by weight of the respirable dry particles.For example, the respirable dry particles of the invention can includeone or more of the salts in a total amount of less than about 45%, lessthan about 40%, less than about 35%, less than about 30%, less thanabout 25%, less than about 20%, less than about 15%, less than about10%, less than about 9%, less than about 8%, less than about 5%, or lessthan about 3% by weight of the respirable dry particles, provided thatthe divalent metal cation is present at less than 3% by weight ofrespirable dry particle.

Alternatively or in addition, the respirable dry particles of theinvention contain a divalent metal cation salt and a monovalent cationsalt, where the divalent cation, as a component of one or more salts, ispresent in an amount of less than 3% by weight of the dry particle, andthe weight ratio of divalent cation to monovalent cation is about 50:1(i.e., about 50 to about 1) to about 0.1:1 (i.e., about 0.1 to about 1).The weight ratio of divalent metal cation to monovalent cation is basedon the amount of divalent metal cation and monovalent cation that arecontained in the divalent metal cation salt and monovalent salts,respectively, that are contained in the dry particle. In particularexamples, the weight ratio of divalent metal cation to monovalent cationis about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1,about 0.7:1, about 0.8:1, about 0.86:1, about 0.92:1, about 1:1; about1.3:1, about 2:1, about 5:1, about 10:1, about 15:1, about 20:1, about25:1, about 30:1, about 35:1, about 40:1, about 45:1, or about 50:1,about 20:1 to about 0.1:1, about 15:1 to about 0.1:1, about 10:1 toabout 0.1:1, or about 5:1 to about 0.1:1.

Alternatively or in addition, the respirable dry particles of theinvention can contain a divalent metal cation salt and a monovalentcation salt, in which the divalent metal cation salt and the monovalentcation salt contain chloride, lactate, citrate or sulfate as the counterion, and the ratio of divalent metal cation (e.g., Ca²⁺, Be²⁺, Mg²⁺,Sr²⁺, Ba²⁺, Fe²⁺) to monovalent cation (e.g, Na⁺, Li⁺, K⁺) mole:mole isabout 50:1 (i.e., about 50 to about 1) to about 0.1:1 (i.e., about 0.1to about 1), provided that the divalent metal cation is present at lessthan 3% by weight of respirable dry particle.

The molar ratio of divalent metal cation to monovalent cation is basedon the amount of divalent metal cation and monovalent cation that arecontained in the divalent metal cation salt and monovalent cation salt,respectively, that are contained in the dry particle. Preferably,divalent metal cation, as a component of one or more divalent metalcation salts, is present in an amount of less than 3% by weight of therespirable dry particle. In particular examples, divalent metal cationand monovalent cation are present in the respirable dry particles in amole ratio of about 8.0:1, about 7.5:1, about 7.0:1, about 6.5:1, about6.0:1, about 5.5:1, about 5.0:1, about 4.5:1, about 4.0:1, about 3.5:1,about 3.0:1, about 2.5:1, about 2.0:1, about 1.5:1, about 1.0:1, about0.77:1, about 0.65:1, about 0.55:1, about 0.45:1, about 0.35:1, about0.25:1, or about 0.2:1, about 8.0:1 to about 0.55:1, about 7.0:1 toabout 0.55:1, about 6.0:1 to about 0.55:1, about 5.0:1 to about 0.55:1,about 4.0:1 to about 0.55:1, about 3.0:1 to about 0.55:1, about 2.0:1 toabout 0.55:1, or about 1.0:1 to about 0.55:1. Alternatively, the ratioof divalent metal cation to monovalent cation is about 8.0:1 to about1:1, about 7.0:1 to about 1:1, about 6.0:1 to about 1:1, about 5.0:1 toabout 1:1, about 4.0:1 to about 1:1, about 4.0:1 to about 2.0:1, about3.9:1 to about 1:1, or about 3.9:1 to about 2.0:1. In a preferredaspect, the ratio of divalent metal cation to monovalent metal cation isfrom about 8:1 to about 2:1, about 4:1 to about 2:1, or 3.9:1 to about2:1.

Preferably, the ratio of divalent metal cation (e.g., Ca²⁺, Be²⁺, Mg²⁺,Sr²⁺, Ba²⁺, Fe²⁺) to monovalent cation (e.g, Na+, Li+, K+) mole:mole isabout 16.0:1.0 to about 1.0:1.0, about 16.0:1.0 to about 2.0:1.0, about8.0:1.0 to about 1.0:1.0, about 4.0:1.0 to about 1.0:1.0, about 4:0:1.0to about 2.0:1.0. More preferably, the divalent metal cation andmonovalent cation are present in the respirable dry particles in a moleratio of about 8.0:1.0 to about 2.0:1.0 or about 4.0:1.0 to about2.0:1.0. Most preferably, the divalent metal cation is Ca²⁺ and themonovalent cation is Na⁺.

If desired, the respirable dry particles described herein can include aphysiologically or pharmaceutically acceptable excipient. For example, apharmaceutically-acceptable excipient includes any of the standardcarbohydrates, sugar alcohols, and amino acids that are known in the artto be useful excipients for inhalation therapy, either alone or in anydesired combination. These excipients are generally relativelyfree-flowing particulates, do not thicken or polymerize upon contactwith water, are toxicologically innocuous when inhaled as a dispersedpowder and do not significantly interact with the active agent in amanner that adversely affects the desired physiological action.

Other suitable excipients can include, for example, sugars (e.g.,lactose, trehalose, maltodextrin), dipalmitoylphosphosphatidylcholine(DPPC), diphosphatidyl glycerol (DPPG),1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS),1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DSPC),1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1-palmitoyl-2-oleoylphosphatidylcholine (POPC), fatty alcohols,polyoxyethylene-9-lauryl ether, surface active fatty, acids, sorbitantrioleate (Span 85), glycocholate, surfactin, poloxomers, sorbitan fattyacid esters, tyloxapol, phospholipids, alkylated sugars, sodiumphosphate, maltodextrin, human serum albumin (e.g., recombinant humanserum albumin), biodegradable polymers (e.g., PLGA), dextran, dextrin,citric acid, sodium citrate, and the like.

Carbohydrate excipients that are useful in this regard include the mono-and polysaccharides. Representative monosaccharides include carbohydrateexcipients such as dextrose (anhydrous and the monohydrate; alsoreferred to as glucose and glucose monohydrate), galactose, mannitol,D-mannose, sorbose and the like. Representative disaccharides includelactose, maltose, sucrose, trehalose and the like. Representativetrisaccharides include raffinose and the like. Other carbohydrateexcipients include maltodextrin and cyclodextrins, such as2-hydroxypropyl-beta-cyclodextrin can be used as desired. Representativesugar alcohols include mannitol, sorbitol and the like.

Suitable amino acid excipients include any of the naturally occurringamino acids that form a powder under standard pharmaceutical processingtechniques and include the non-polar (hydrophobic) amino acids and polar(uncharged, positively charged and negatively charged) amino acids, suchamino acids are of pharmaceutical grade and are generally regarded assafe (GRAS) by the U.S. Food and Drug Administration. Representativeexamples of non-polar amino acids include alanine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan and valine.Representative examples of polar, uncharged amino acids include cystine,glycine, glutamine, serine, threonine, and tyrosine. Representativeexamples of polar, positively charged amino acids include arginine,histidine and lysine. Representative examples of negatively chargedamino acids include aspartic acid and glutamic acid. These amino acidsare generally available from commercial sources that providepharmaceutical-grade products such as the Aldrich Chemical Company,Inc., Milwaukee, Wis. or Sigma Chemical Company, St. Louis, Mo.

Preferably, the excipients are chosen from one or more of the following;sugars (e.g., lactose, trehalose), polysaccharide (e.g., dextrin,maltodextrin, dextran, raffinose), sugar alcohols (e.g., mannitol,xylitol, sorbitol), and amino acids (e.g., glycine, alanine, leucine,isoleucine). More preferably, the excipients are chosen from one or moreof the following: leucine, mannitol, and maltodextrin. In one aspect ofthe invention, the excipient is not a phospholipid, e.g.,dipalmitoylphosphosphatidylcholine (DPPC), diphosphatidyl glycerol(DPPG), 1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS),1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DSPC),1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1-palmitoyl-2-oleoylphosphatidylcholine (POPC). In another aspect of theinvention, the excipient is not a carboxylate acid or its salt form,e.g., citric acid or sodium citrate.

Preferred amino acid excipients, such as the hydrophobic amino acidleucine, can be present in the dry particles of the invention in anamount of about 99% or less by weight of respirable dry particles. Forexample, the respirable dry particles of the invention can contain theamino acid leucine in an amount of about 0.1% to about 10% by weight, 5%to about 30% by weight, about 10% to about 20% by weight, about 5% toabout 20% by weight, about 11% to about 50% by weight, about 15% toabout 50% by weight, about 20% to about 50% by weight, about 30% toabout 50% by weight, about 11% to about 40% by weight, about 11% toabout 30% by weight, about 11% to about 20% by weight, about 20% toabout 40% by weight, about 51% to about 99% by weight, about 60% toabout 99% by weight, about 70% to about 99% by weight, about 80% toabout 99% by weight, about 51% to about 90% by weight, about 51% toabout 80% by weight, about 51% to about 70% by weight, about 60% toabout 90% by weight, about 70% to about 90% by weight, about 45% or lessby weight, about 40% or less by weight, about 35% or less by weight,about 30% or less by weight, about 25% or less by weight, about 20% orless by weight, about 18% or less by weight, about 16% or less byweight, about 15% or less by weight, about 14% or less by weight, about13% or less by weight, about 12% or less by weight, about 11% or less byweight, about 10% or less by weight, about 9% or less by weight, about8% or less by weight, about 7% or less by weight, about 6% or less byweight, about 5% or less by weight, about 4% or less by weight, about 3%or less by weight, about 2% or less by weight, or about 1% or less byweight.

Preferred carbohydrate excipients, such as maltodextrin and mannitol,can be present in the dry particles of the invention in an amount ofabout 99% or less by weight of respirable dry particles. For example,the respirable dry particles of the invention can contain maltodextrinin an amount of about 0.1% to about 10% by weight, 5% to about 30% byweight by weight, about 10% to about 20% by weight by weight, about 5%to about 20% by weight, about 11% to about 50% by weight, about 15% toabout 50% by weight, about 20% to about 50% by weight, about 30% toabout 50% by weight, about 11% to about 40% by weight, about 11% toabout 30% by weight, about 11% to about 20% by weight, about 20% toabout 40% by weight, about 51% to about 99% by weight, about 60% toabout 99% by weight, about 70% to about 99% by weight, about 80% toabout 99% by weight, about 51% to about 90% by weight, about 51% toabout 80% by weight, about 51% to about 70% by weight, about 60% toabout 90% by weight, about 70% to about 90% by weight, about 45% or lessby weight, about 40% or less by weight, about 35% or less by weight,about 30% or less by weight, about 25% or less by weight, about 20% orless by weight, about 18% or less by weight, about 16% or less byweight, about 15% or less by weight, about 14% or less by weight, about13% or less by weight, about 12% or less by weight, about 11% or less byweight, about 10% or less by weight, about 9% or less by weight, about8% or less by weight, about 7% or less by weight, about 6% or less byweight, about 5% or less by weight, about 4% or less by weight, about 3%or less by weight, about 2% or less by weight, or about 1% or less byweight.

In some preferred aspects, the dry particles contain an excipientselected from leucine, maltodextrin, mannitol and any combinationthereof. In particular embodiments, the excipient is leucine,maltodextrin, or mannitol.

If desired, the dry particles or dry powders described herein caninclude one or more additional active agents, such as mucoactive ormucolytic agents, surfactants, antibiotics, antivirals, antihistamines,cough suppressants, bronchodilators, anti-inflammatory agents, steroids,vaccines, adjuvants, expectorants, macromolecules, or therapeutics thatare helpful for chronic maintenance of cystic fibrosis (CF). Preferredactive agents include, but are not limited to, LABAs (e.g., formoterol,salmeterol), short-acting beta agonists (e.g., albuterol),corticosteroids (e.g., fluticasone), LAMAs (e.g., tiotropium),antibiotics (e.g., levofloxacin, tobramycin), antibodies (e.g.,therapeutic antibodies), hormones (e.g., insulin), cytokines, growthfactors, and combinations thereof. When the dry powders are intended fortreatment of CF, preferred additional active agents are short-actingbeta agonists (e.g., albuterol), antibiotics (e.g., levofloxacin),recombinant human deoxyribonuclease I (e.g., dornase alfa, also known asDNase), sodium channel blockers (e.g., amiloride), and combinationsthereof.

In some embodiments, the dry particles or dry powders described hereincan contain an agent that disrupts and/or disperses biofilms. Suitableexamples of agents to promote disruption and/or dispersion of biofilmsinclude specific amino acid stereoisomers, e.g., D-leucine,D-methionine, D-tyrosine, D-tryptophan, and the like. (Kolodkin-Gal, I.,D. Romero, et al. “D-amino acids trigger biofilm disassembly.” Science328(5978): 627-629.) For example, all or a portion of the leucine in thedry powders described herein which contain leucine can be D-leucine.

Examples of suitable mucoactive or mucolytic agents include MUC5AC andMUC5B mucins, DNase, N-acetylcysteine (NAC), cysteine, nacystelyn,dornase alfa, gelsolin, heparin, heparin sulfate, P2Y2 agonists (e.g.,UTP, INS365), nedocromil sodium, hypertonic saline, and mannitol.

Suitable surfactants include L-alpha-phosphatidylcholine dipalmitoyl(“DPPC”), diphosphatidyl glycerol (DPPG),1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS),1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DSPC),1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1-palmitoyl-2-oleoylphosphatidylcholine (POPC), fatty alcohols,polyoxyethylene-9-lauryl ether, surface active fatty, acids, sorbitantrioleate (Span 85), glycocholate, surfactin, poloxomers, sorbitan fattyacid esters, tyloxapol, phospholipids, and alkylated sugars.

If desired, the dry particles or dry powders described herein cancontain an antibiotic. The antibiotic can be suitable for treating anydesired bacterial infection. Dry particles or dry powders that containan antibiotic can be used to reduce the spread of infection, eitherwithin a patient or from patient to patient. For example, dry particlesor dry powders s for treating bacterial pneumonia or VAT, can furthercomprise an antibiotic, such as a macrolide (e.g., azithromycin,clarithromycin and erythromycin), a tetracycline (e.g., doxycycline,tigecycline), a fluoroquinolone (e.g., gemifloxacin, levofloxacin,ciprofloxacin and mocifloxacin), a cephalosporin (e.g., ceftriaxone,defotaxime, ceftazidime, cefepime), a penicillin (e.g., amoxicillin,amoxicillin with clavulanate, ampicillin, piperacillin, and ticarcillin)optionally with a β-lactamase inhibitor (e.g., sulbactam, tazobactam andclavulanic acid), such as ampicillin-sulbactam, piperacillin-tazobactamand ticarcillin with clavulanate, an aminoglycoside (e.g., amikacin,arbekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodostreptomycin, streptomycin, tobramycin, and apramycin), a penem orcarbapenem (e.g., doripenem, ertapenem, imipenem and meropenem), amonobactam (e.g., aztreonam), an oxazolidinone (e.g., linezolid),vancomycin, glycopeptide antibiotics (e.g., telavancin),tuberculosis-mycobacterium antibiotics and the like.

If desired, the dry particles or dry powders described herein cancontain an agent for treating infections with mycobacteria, such asMycobacterium tuberculosis. Suitable agents for treating infections withmycobacteria (e.g., M. tuberculosis) include an aminoglycoside (e.g.,capreomycin, kanamycin, streptomycin), a fluoroquinolone (e.g.,ciprofloxacin, levofloxacin, moxifloxacin), isozianid and isozianidanalogs (e.g., ethionamide), aminosalicylate, cycloserine,diarylquinoline, ethambutol, pyrazinamide, protionamide, rifampin, andthe like.

If desired, the dry particles or dry powders described herein cancontain a suitable antiviral agent, such as oseltamivir, zanamavir,amantidine, rimantadine, ribavirin, gancyclovir, valgancyclovir,foscavir, Cytogam® (Cytomegalovirus Immune Globulin), pleconaril,rupintrivir, palivizumab, motavizumab, cytarabine, docosanol, denotivir,cidofovir, and acyclovir. The dry particles or dry powders can contain asuitable anti-influenza agent, such as zanamivir, oseltamivir,amantadine, or rimantadine.

Suitable antihistamines include clemastine, asalastine, loratadine,fexofenadine and the like.

Suitable cough suppressants include benzonatate, benproperine,clobutinal, diphenhydramine, dextromethorphan, dibunate, fedrilate,glaucine, oxalamine, piperidione, opiods such as codeine and the like.

Suitable brochodilators include short-acting beta2 agonists, long-actingbeta2 agonists (LABA), long-acting muscarinic anagonists (LAMA),combinations of LABAs and LAMAs, methylxanthines, short-actinganticholinergic agents (may also be referred to as short actinganti-muscarinic), long-acting bronchodilators and the like.

Suitable short-acting beta₂ agonists include albuterol, epinephrine,pirbuterol, levalbuterol, metaproteronol, maxair, and the like.

Examples of albuterol sulfate formulations (also called salbutamol)include Inspiryl (AstraZeneca Plc), Salbutamol SANDOZ (Sanofi-Aventis),Asmasal clickhaler (Vectura Group Plc.), Ventolin® (GlaxoSmithKlinePlc), Salbutamol GLAND (GlaxoSmithKline Plc), Airomir® (TevaPharmaceutical Industries Ltd.), ProAir HFA (Teva PharmaceuticalIndustries Ltd.), Salamol (Teva Pharmaceutical Industries Ltd.), Ipramol(Teva Pharmaceutical Industries Ltd), Albuterol sulfate TEVA (TevaPharmaceutical Industries Ltd), and the like. Examples of epinephrineinclude Epinephine Mist KING (King Pharmaceuticals, Inc.), and the like.Examples of pirbuterol as pirbuterol acetate include Maxair® (TevaPharmaceutical Industries Ltd.), and the like. Examples of levalbuterolinclude Xopenex® (Sepracor), and the like. Examples of metaproteronolformulations as metaproteronol sulfate include Alupent® (BoehringerIngelheim GmbH), and the like.

Suitable LABAs include salmeterol, formoterol and isomers (e.g.,arformoterol), clenbuterol, tulobuterol, vilanterol (Revolair™),indacaterol, carmoterol, isoproterenol, procaterol, bambuterol,milveterol, olodaterol, and the like.

Examples of salmeterol formulations include salmeterol xinafoate asSerevent® (GlaxoSmithKline Plc), salmeterol as Inaspir (LaboratoriosAlmirall, S.A.), Advair® HFA (GlaxoSmithKline PLC), Advair Diskus®(GlaxoSmithKline PLC, Theravance Inc), Plusvent (Laboratorios Almirall,S.A.), VR315 (Novartis, Vectura Group PLC) and the like. Examples offormoterol and isomers (e.g., arformoterol) include Foster (ChiesiFarmaceutici S.p.A), Atimos (Chiesi Farmaceutici S.p.A, NycomedInternaional Management), Flutiform® (Abbott Laboratories, SkyePharmaPLC), MFF258 (Novartis AG), Formoterol clickhaler (Vectura Group PLC),Formoterol HFA (SkyePharma PLC), Oxis® (Astrazeneca PLC), Oxis pMDI(Astrazeneca), Foradil® Aerolizer (Novartis, Schering-Plough Corp,Merck), Foradil® Certihaler (Novartis, SkyePharma PLC), Symbicort®(AstraZeneca), VR632 (Novartis AG, Sandoz International GmbH), MFF258(Merck & Co Inc, Novartis AG), Alvesco® Combo (Nycomed InternationalManagement GmbH, Sanofi-Aventis, Sepracor Inc), Mometasone furoate(Schering-Plough Corp), and the like. Examples of clenbuterol includeVentipulmin® (Boehringer Ingelheim), and the like. Examples oftulobuterol include Hokunalin Tape (Abbott Japan Co., Ltd., Maruho Co.,Ltd.), and the like. Examples of vilanterol include Revolair™(GlaxoSmithKline PLC), GSK64244 (GlaxoSmithKline PLC), and the like.Examples of indacaterol include QAB 149 (Novartis AG, SkyePharma PLC),QMF149 (Merck & Co Inc) and the like. Examples of carmoterol includeCHF4226 (Chiese Farmaceutici S.p.A., Mitsubishi Tanabe PharmaCorporation), CHF5188 (Chiesi Farmaceutici S.p.A), and the like.Examples of isoproterenol sulfate include Aludrin (Boehringer IngelheimGmbH) and the like. Examples of procaterol include Meptin clickhaler(Vectura Group PLC), and the like. Examples of bambuterol include Bambec(AstraZeneca PLC), and the like. Examples of milveterol includeGSK159797C (GlaxoSmithKline PLC), TD3327 (Theravance Inc), and the like.Examples of olodaterol include BI1744CL (Boehringer Ingelheim GmbH) andthe like.

Examples of LAMAs include tiotroprium (Spiriva), trospium chloride,glycopyrrolate, aclidinium, ipratropium and the like.

Examples of tiotroprium formulations include Spiriva®(Boehringer-Ingleheim, Pfizer), and the like. Examples of glycopyrrolateinclude Robinul® (Wyeth-Ayerst), Robinul® Forte (Wyeth-Ayerst), NVA237(Novartis), and the like. Examples of aclidinium include Eklira (ForestLabaoratories, Almirall), and the like.

Examples of combinations of LABAs and LAMAs include indacaterol withglycopyrrolate, formoterol with glycopyrrolate, indacaterol withtiotropium, olodaterol and tiotropium, vilanterol with a LAMA, and thelike. Examples of combinations of formoterol with glycopyrrolate includePT003 (Pearl Therapeutics) and the like. Examples of combinations ofolodaterol with tiotropium include BI1744 with Spirva (BoehringerIngelheim) and the like. Examples of combinations of vilanterol with aLAMA include GSK573719 with GSK642444 (GlaxoSmithKline PLC), and thelike.

Examples of combinations of indacaterol with glycopyrrolate includeQVA149A (Novartis), and the like.

Examples of methylxanthine include aminophylline, ephedrine,theophylline, oxtriphylline, and the like.

Examples of aminophylline formulations include Aminophylline BOEHRINGER(Boehringer Ingelheim GmbH) and the like. Examples of ephedrine includeBronkaid® (Bayer AG), Broncholate (Sanofi-Aventis), Primatene® (Wyeth),Tedral SA®, Marax (Pfizer Inc) and the like. Examples of theophyllineinclude Euphyllin (Nycomed International Management GmbH), Theo-dur(Pfizer Inc, Teva Pharmacetuical Industries Ltd) and the like. Examplesof oxtriphylline include Choledyl SA (Pfizer Inc) and the like.

Examples of short-acting anticholinergic agents include ipratropiumbromide, and oxitropium bromide.

Examples of ipratropium bromide formulations includeAtrovent®/Apovent/Inpratropio (Boehringer Ingelheim GmbH), Ipramol (TevaPharmaceutical Industries Ltd) and the like. Examples of oxitropiumbromide include Oxivent (Boehringer Ingelheim GmbH), and the like.

Suitable anti-inflammatory agents include leukotriene inhibitors,phosphodiesterase 4 (PDE4) inhibitors, other anti-inflammatory agents,and the like.

Suitable leukotriene inhibitors include montelukast formulations(cystinyl leukotriene inhibitors), masilukast, zafirleukast (leukotrieneD4 and E4 receptor inhibitors), pranlukast, zileuton (5-lipoxygenaseinhibitors), and the like.

Examples of montelukast (cystinyl leukotriene inhibitor) includeSingulair® (Merck & Co Inc), Loratadine, montelukast sodium SCHERING(Schering-Plough Corp), MK0476C (Merck & Co Inc), and the like. Examplesof masilukast include MCC847 (AstraZeneca PLC), and the like. Examplesof zafirlukast (leukotriene D4 and E4 receptor inhibitor) includeAccolate® (AstraZeneca PLC), and the like. Examples of pranlukastinclude Azlaire (Schering-Plough Corp). Examples of zileuton (5-LO)include Zyflo® (Abbott Laboratories), Zyflo CR® (Abbott Laboratories,SkyePharma PLC), Zileuton ABBOTT LABS (Abbott Laboratories), and thelike. Suitable PDE4 inhibitors include cilomilast, roflumilast,oglemilast, tofimilast, and the like.

Examples of cilomilast formulations include Ariflo (GlaxoSmithKlinePLC), and the like. Examples of roflumilast include Daxas® (NycomedInternational Management GmbH, Pfizer Inc), APTA2217 (Mitsubishi TanabePharma Corporation), and the like. Examples of oglemilast includeGRC3886 (Forest Laboratories Inc), and the like. Examples of tofimilastinclude Tofimilast PFIZER INC (Pfizer Inc), and the like.

Other anti-inflammatory agents include omalizumab (anti-IgEimmunoglobulin Daiichi Sankyo Company, Limited), Zolair (anti-IgEimmunoglobulin, Genentech Inc, Novartis AG, Roche Holding Ltd), Solfa(LTD4 antagonist and phosphodiesterase inhibitor, Takeda PharmaceuticalCompany Limited), IL-13 and IL-13 receptor inhibitors (such as AMG-317,MILR1444A, CAT-354, QAX576, IMA-638, Anrukinzumab, IMA-026, MK-6105,DOM-0910, and the like), IL-4 and IL-4 receptor inhibitors (such asPitrakinra, AER-003, AIR-645, APG-201, DOM-0919, and the like), IL-1inhibitors such as canakinumab, CRTh2 receptor antagonists such asAZD1981 (CRTh2 receptor antagonist, AstraZeneca), neutrophil elastaseinhibitor such as AZD9668 (neutrophil elastase inhibitor, fromAstraZeneca), GW856553X Losmapimod (P38 kinase inhibitor,GlaxoSmithKline PLC), Arofylline LAB ALMIRALL (PDE-4 inhibitor,Laboratorios Almirall, S.A.), ABT761 (5-LO inhibitor, AbbottLaboratories), Zyflo® (5-LO inhibitor, Abbott Laboratories), BT061(anti-CD4 mAb, Boehringer Ingelheim GmbH), Corns (inhaled lidocaine todecrease eosinophils, Gilead Sciences Inc), Prograf (IL-2-mediatedT-cell activation inhibitor, Astellas Pharma), Bimosiamose PFIZER INC(selectin inhibitor, Pfizer Inc), R411 (α4 β1/α4 β7 integrin antagonist,Roche Holdings Ltd), Tilade® (inflammatory mediator inhibitor,Sanofi-Aventis), Orenica® (T-cell co-stimulation inhibitor,Bristol-Myers Squibb Company), Soliris® (anti-CS, AlexionPharmaceuticals Inc), Entorken® (Farmacija d.o.o.), Excellair® (Sykkinase siRNA, ZaBeCor Pharmaceuticals, Baxter International Inc), KB003(anti-GMCSF mAb, KaloBios Pharmaceuticals), Cromolyn sodiums (inhibitrelease of mast cell mediators): Cromolyn sodium BOEHRINGER (BoehringerIngelheim GmbH), Cromolyn sodium TEVA (Teva Pharmaceutical IndustriesLtd), Intal (Sanofi-Aventis), BI1744CL (oldaterol (β2-adrenoceptorantagonist) and tiotropium, Boehringer Ingelheim GmbH), NFκ-Binhibitors, CXR2 antagaonists, HLE inhibitors, HMG-CoA reductaseinhibitors and the like.

Anti-inflammatory agents also include compounds that inhibit/decreasecell signaling by inflammatory molecules like cytokines (e.g., IL-1,IL-4, IL-5, IL-6, IL-9, IL-13, IL-18 IL-25, IFN-α, IFN-β, and others),CC chemokines CCL-1-CCL28 (some of which are also known as, for example,MCP-1, CCL2, RANTES), CXC chemokines CXCL1-CXCL17 (some of which arealso know as, for example, IL-8, MIP-2), growth factors (e.g., GM-CSF,NGF, SCF, TGF-β, EGF, VEGF and others) and/or their respectivereceptors.

Some examples of the aforementioned anti-inflammatoryantagonists/inhibitors include ABN912 (MCP-1/CCL2, Novartis AG), AMG761(CCR4, Amgen Inc), Enbrel® (TNF, Amgen Inc, Wyeth), huMAb OX40LGENENTECH (TNF superfamily, Genentech Inc, AstraZeneca PLC), R4930 (TNFsuperfamily, Roche Holding Ltd), SB683699/Firategrast (VLA4,GlaxoSmithKline PLC), CNT0148 (TNFα, Centocor, Inc, Johnson & Johnson,Schering-Plough Corp); Canakinumab (IL-1β, Novartis); IsrapafantMITSUBISHI (PAF/IL-5, Mitsubishi Tanabe Pharma Corporation); IL-4 andIL-4 receptor antagonists/inhibitors: AMG317 (Amgen Inc), BAY169996(Bayer AG), AER-003 (Aerovance), APG-201 (Apogenix); IL-5 and IL-5receptor antagonists/inhibitors: MEDI563 (AstraZeneca PLC, MedImmune,Inc), Bosatria® (GlaxoSmithKline PLC), Cinquil® (Ception Therapeutic),TMC120B (Mitsubishi Tanabe Pharma Corporation), Bosatria(GlaxoSmithKline PLC), Reslizumab SCHERING (Schering-Plough Corp);MEDI528 (IL-9, AstraZeneca, MedImmune, Inc); IL-13 and IL-13 receptorantagonists/inhibitors: TNX650 GENENTECH (Genentech), CAT-354(AstraZeneca PLC, MedImmune), AMG-317 (Takeda Pharmaceutical CompanyLimited), MK6105 (Merck & Co Inc), IMA-026 (Wyeth), IMA-638 Anrukinzumab(Wyeth), MILR1444A/Lebrikizumab (Genentech), QAX576 (Novartis), CNTO-607(Centocor), MK-6105 (Merck, CSL); Dual IL-4 and IL-13 inhibitors:AIR645/ISIS369645 (ISIS Altair), DOM-0910 (GlaxoSmithKline, Domantis),Pitrakinra/AER001/Aerovant™ (Aerovance Inc), AMG-317 (Amgen), and thelike.

Suitable steroids include corticosteroids, combinations ofcorticosteroids and LABAs, combinations of corticosteroids and LAMAs,combinations of corticosteroids, LABAs and LAMAs, and the like.

Suitable corticosteroids include budesonide, fluticasone, flunisolide,triamcinolone, beclomethasone, mometasone, ciclesonide, dexamethasone,and the like.

Examples of budesonide formulations include Captisol-Enabled® BudesonideSolution for Nebulization (AstraZeneca PLC), Pulmicort®(AstraZenecaPLC), Pulmicort® Flexhaler (AstraZeneca Plc), Pulmicort® HFA-MDI(AstraZeneca PLC), Pulmicort Respules® (AstraZeneca PLC), Inflammide(Boehringer Ingelheim GmbH), Pulmicort® HFA-MDI (SkyePharma PLC), UnitDose Budesonide ASTRAZENECA (AstraZeneca PLC), Budesonide Modulite(Chiesi Farmaceutici S.p.A), CHF5188 (Chiesi Farmaceutici S.p.A),Budesonide ABBOTT LABS (Abbott Laboratories), Budesonide clickhaler(Vestura Group PLC), Miflonide (Novartis AG), Xavin (Teva PharmaceuticalIndustries Ltd.), Budesonide TEVA (Teva Pharmaceutical Industries Ltd.),Symbicort® (AstraZeneca K.K., AstraZeneca PLC), VR632 (Novartis AG,Sandoz International GmbH), and the like.

Examples of fluticasone propionate formulations include FlixotideEvohaler (GlaxoSmithKline PLC), Flixotide Nebules (GlaxoSmithKline Plc),Flovent® (GlaxoSmithKline Plc), Flovent® Diskus (GlaxoSmithKline PLC),Flovent® HFA (GlaxoSmithKline PLC), Flovent® Rotadisk (GlaxoSmithKlinePLC), Advair® HFA (GlaxoSmithKline PLC, Theravance Inc), Advair Diskus®(GlaxoSmithKline PLC, Theravance Inc.), VR315 (Novartis AG, VecturaGroup PLC, Sandoz International GmbH), and the like. Other formulationsof fluticasone include fluticasone as Flusonal (Laboratorios Almirall,S.A.), fluticasone furoate as GW685698 (GlaxoSmithKline PLC, ThervanceInc.), Plusvent (Laboratorios Almirall, S.A.), Flutiform® (AbbottLaboratories, SkyePharma PLC), and the like.

Examples of flunisolide formulations include Aerobid® (ForestLaboratories Inc), Aerospan® (Forest Laboratories Inc), and the like.Examples of triamcinolone include Triamcinolone ABBOTT LABS (AbbottLaboratories), Azmacort® (Abbott Laboratories, Sanofi-Aventis), and thelike. Examples of beclomethasone dipropionate include Beclovent(GlaxoSmithKline PLC), QVAR® (Johnson & Johnson, Schering-Plough Corp,Teva Pharmacetucial Industries Ltd), Asmabec clickhaler (Vectura GroupPLC), Beclomethasone TEVA (Teva Pharmaceutical Industries Ltd), Vanceril(Schering-Plough Corp), BDP Modulite (Chiesi Farmaceutici S.p.A.),Clenil (Chiesi Farmaceutici S.p.A), Beclomethasone dipropionate TEVA(Teva Pharmaceutical Industries Ltd), and the like. Examples ofmometasone include QAB 149 Mometasone furoate (Schering-Plough Corp),QMF149 (Novartis AG), Fomoterol fumarate, mometoasone furoate(Schering-Plough Corp), MFF258 (Novartis AG, Merck & Co Inc), Asmanex®Twisthaler (Schering-Plough Corp), and the like. Examples of cirlesonideinclude Alvesco® (Nycomed International Management GmbH, Sepracor,Sanofi-Aventis, Tejin Pharma Limited), Alvesco® Combo (NycomedInternational Management GmbH, Sanofi-Aventis), Alvesco® HFA (NycomedIntenational Management GmbH, Sepracor Inc), and the like. Examples ofdexamethasone include DexPak® (Merck), Decadron® (Merck), Adrenocot,CPC-Cort-D, Decaject-10, Solurex and the like. Other corticosteroidsinclude Etiprednol dicloacetate TEVA (Teva Pharmaceutical IndustriesLtd), and the like.

Combinations of corticosteroids and LABAs include salmeterol withfluticasone, formoterol with budesonide, formoterol with fluticasone,formoterol with mometasone, indacaterol with mometasone, and the like.

Examples of salmeterol with fluticasone include Plusvent (LaboratoriosAlmirall, S.A.), Advair® HFA (GlaxoSmithKline PLC), Advair® Diskus(GlaxoSmithKline PLV, Theravance Inc), VR315 (Novartis AG, Vectura GroupPLC, Sandoz International GmbH) and the like. Examples of formoterolwith budesonide include Symbicort® (AstraZeneca PLC), VR632 (NovartisAG, Vectura Group PLC), and the like. Examples of vilanterol withfluticasone include GSK642444 with fluticasone and the like. Examples offormoterol with fluticasone include Flutiform® (Abbott Laboratories,SkyePharma PLC), and the like. Examples of formoterol with mometasoneinclude Dulera®/MFF258 (Novartis AG, Merck & Co Inc), and the like.Examples of indacaterol with mometasone include QAB149 Mometasonefuroate (Schering-Plough Corp), QMF149 (Novartis AG), and the like.Combinations of corticosteroids with LAMAs include fluticasone withtiotropium, budesonide with tiotropium, mometasone with tiotropium,salmeterol with tiotropium, formoterol with tiotropium, indacaterol withtiotropium, vilanterol with tiotropium, and the like. Combinations ofcorticosteroids with LAMAs and LABAs include, for example, fluticasonewith salmeterol and tiotropium.

Other anti-asthma molecules include: ARD111421 (VIP agonist, AstraZenecaPLC), AVE0547 (anti-inflammatory, Sanofi-Aventis), AVE0675 (TLR agonist,Pfizer, Sanofi-Aventis), AVE0950 (Syk inhibitor, Sanofi-Aventis),AVE5883 (NK1/NK2 antagonist, Sanofi-Aventis), AVE8923 (tryptase betainhibitor, Sanofi-Aventis), CGS21680 (adenosine A2A receptor agonist,Novartis AG), ATL844 (A2B receptor antagonist, Novartis AG), BAY443428(tryptase inhibitor, Bayer AG), CHF5407 (M3 receptor inhibitor, ChiesiFarmaceutici S.p.A.), CPLA2 Inhibitor WYETH (CPLA2 inhibitor, Wyeth),IMA-638 (IL-13 antagonist, Wyeth), LAS100977 (LABA, LaboratoriosAlmirall, S.A.), MABA (M3 and β2 receptor antagonist, ChiesiFarmaceutici S.p.A), R1671 (mAb, Roche Holding Ltd), CS003 (Neurokininreceptor antagonist, Daiichi Sankyo Company, Limited), DPC168 (CCRantagonist, Bristol-Myers Squibb), E26 (anti-IgE, Genentech Inc), HAE1(Genentech), IgE inhibitor AMGEN (Amgen Inc), AMG853 (CRTH2 and D2receptor antagonist, Amgen), IPL576092 (LSAID, Sanofi-Aventis), EPI2010(antisense adenosine 1, Chiesi Farmaceutici S.p.A.), CHF5480 (PDE-4inhibitor, Chiesi Farmaceutici S.p.A.), KI04204 (corticosteroid, AbbottLaboratories), SVT47060 (Laboratorios Salvat, S.A.), VML530 (leukotrienesynthesis inhibitor, Abbott Laboratories), LAS35201 (M3 receptorantagonist, Laboratorios Almirall, S.A.), MCC847 (D4 receptorantagonist, Mitsubishi Tanabe Pharma Corporation), MEM1414 (PDE-4inhibitor, Roche), TA270 (5-LO inhibitor, Chugai Pharmaceutical Co Ltd),TAK661 (eosinophil chemotaxis inhibitor, Takeda Pharmaceutical CompanyLimited), TBC4746 (VLA-4 antagonist, Schering-Plough Corp), VR694(Vectura Group PLC), PLD177 (steroid, Vectura Group PLC), KI03219(corticosteroid+LABA, Abbott Laboratories), AMG009 (Amgen Inc), AMG853(D2 receptor antagonist, Amgen Inc);

AstraZeneca PLC: AZD1744 (CCR3/histamine-1 receptor antagonist, AZD1419(TLR9 agonist), Mast Cell inhibitor ASTRAZENECA, AZD3778 (CCRantagonist), DSP3025 (TLR7 agonist), AZD1981 (CRTh2 receptorantagonist), AZD5985 (CRTh2 antagonist), AZD8075 (CRTh2 antagonist),AZD1678, AZD2098, AZD2392, AZD3825 AZD8848, AZD9215, ZD2138 (5-LOinhibitor), AZD3199 (LABA);

GlaxoSmithKline PLC: GW328267 (adenosine A2 receptor agonist), GW559090(α4 integrin antagonist), GSK679586 (mAb), GSK597901 (adrenergic β2agonist), AM103 (5-LO inhibitor), GSK256006 (PDE4 inhibitor), GW842470(PDE-4 inhibitor), GSK870086 (glucocorticoid agonist), GSK159802 (LABA),GSK256066 (PDE-4 inhibitor), GSK642444 (LABA, adrenergic β2 agonist),GSK64244 and Revolair (fluticasone/vilanterol), GSK799943(corticosteroid), GSK573719 (mAchR antagonist), and GSK573719;

Pfizer Inc: PF3526299, PF3893787, PF4191834 (FLAP antagonist), PF610355(adrenergic β2 agonist), CP664511 (α4β1/VCAM-1 interaction inhibitor),CP609643 (inhibitor of α4β1/VCAM-1 interactions), CP690550 (JAK3inhibitor), SAR21609 (TLR9 agonist), AVE7279 (Th1 switching), TBC4746(VLA-4 antagonist); R343 (IgE receptor signaling inhibitor), SEP42960(adenosine A3 antagonist);

Sanofi-Aventis: MLN6095 (CrTH2 inhibitor), SAR137272 (Az3 antagonist),SAR21609 (TLR9 agonist), SAR389644 (DP1 receptor antagonist), SAR398171(CRTH2 antagonist), SSR161421 (adenosine A3 receptor antagonist);

Merck & Co Inc: MK0633, MK0633, MK0591 (5-LO inhibitor), MK886(leukotriene inhibitor), BIO1211 (VLA-4 antagonist); Novartis AG: QAE397(long-acting corticosteroid), QAK423, QAN747, QAP642 (CCR3 antagonist),QAX935 (TLR9 agonist), NVA237 (LAMA).

Suitable expectorants include guaifenesin, guaiacolculfonate, ammoniumchloride, potassium iodide, tyloxapol, antimony pentasulfide and thelike.

Suitable vaccines include nasally inhaled influenza vaccines and thelike.

The active agent can also be selected from the group consisting oftransient receptor potential (TRP) channel agonists. In certainembodiments, the TRP agonist is a TRPC, TRPV, TRPM and/or TRPA1subfamily agonist. In some embodiments, the TRP channel agonist isselected from the group consisting of TRPV2, TRPV3, TRPV4, TRPC6, TRPM6,and/or TRPA1 agonist. Suitable TRP channel agonists may be selected fromthe group consisting of allyl isothiocyanate (AITC), benyzlisothiocyanate (BITC), phenyl isothiocyanate, isopropyl isothiocyanate,methyl isothiocyanate, diallyl disulfide, acrolein (2-propenal),disulfuram (Antabuse®), farnesyl thiosalicylic acid (FTS), farnesylthioacetic acid (FTA), chlodantoin (Sporostacin®, topical fungicidal),(15-d-PGJ2), 5,8,11,14 eicosatetraynoic acid (ETYA), dibenzoazepine,mefenamic acid, fluribiprofen, keoprofen, diclofenac, indomethacin, SCalkyne (SCA), pentenal, mustard oil alkyne (MOA), iodoacetamine,iodoacetamide alkyne, (2-aminoethyl) methanethiosulphonate (MTSEA),4-hydroxy-2-noneal (HNE), 4-hydroxy xexenal (HHE),2-chlorobenzalmalononitrile, N-chloro tosylamide (chloramine-T),formaldehyde, isoflurane, isovelleral, hydrogen peroxide, URB597,thiosulfinate, Allicin (a specific thiosulfinate), flufenamic acid,niflumic acid, carvacrol, eugenol, menthol, gingerol, icilin, methylsalicylate, arachidonic acid, cinnemaldehyde, super sinnemaldehyde,tetrahydrocannabinol (THC or Δ⁹-THC), cannabidiol (CBD), cannabichromene(CBC), cannabigerol (CBG), THC acid (THC-A), CBD acid (CBD-A), CompoundI (AMG5445),4-methyl-N-[2,2,2-trichloro-1-(4-chlorophenylsulfanyl)ethyl]benzamide,N-[2,2,2-trichloro-1-(4-chlorophenylsulfanyl)ethyl]acetamid, AMG9090,AMG5445, 1-oleoyl-2-acetyl-sn-glycerol (OAG), carbachol, diacylglycerol(DAG), 1,2-Didecanoylglycerol, flufenamate/flufenamic acid,niflumate/niflumic acid, hyperforin, 2-aminoethoxydiphenyl borate(2-APB), diphenylborinic anhydride (DPBA), delta-9-tetrahydrocannabinol(Δ⁹-THC or THC), cannabiniol (CBN), 2-APB, O-1821,11-hydroxy-Δ9-tetrahydrocannabinol, nabilone, CP55940, HU-210,HU-211/dexanabinol, HU-331, HU-308, JWH-015,WIN55,212-2,2-Arachidonoylglycerol (2-AG), Arvil, PEA, AM404, O-1918,JWH-133, incensole, incensole acetate, menthol, eugenol, dihydrocarveol,carveol, thymol, vanillin, ethyl vanillin, cinnemaldehyde, 2aminoethoxydiphenyl borate (2-APB), diphenylamine (DPA), diphenylborinicanhydride (DPBA), camphor, (+)-borneol, (−)-isopinocampheol,(−)-fenchone, (−)-trans-pinocarveol, isoborneol, (—O-camphorquinone,(−)-α-thujone, α-pinene oxide, 1,8-cineole/eucalyptol, 6-butyl-m-cresol,carvacrol, p-sylenol, kreosol, propofol, p-cymene, (−)-isoppulegol,(−)-carvone, (+)-dihydrocarvone, (−)-menthone, (+)-linalool, geraniol,1-isopropyl-4-methylbicyclo[3.1.0]hexan-4-ol, 4αPDD, GSK1016790A,5′6′Epoxyeicosatrienoic (5′6′-EET), 8′9′Epoxyeicosatrienoic (8′9′-EET),APP44-1, RN1747, Formulation Ib WO200602909, Formulation IIbWO200602909, Formulation IIc WO200602929, Formulation IId WO200602929,Formulation IIb WO200602929, Formulation IIIc WO200602929, arachidonicacid (AA), 12-O-Tetradecanoylphorbol-13-acetate (TPA)/phorbol12-myristate 13-acetate (PMA), bisandrographalide (BAA), incensole,incensole acetate, Compound IX WO2010015965, Compound X WO2010015965,Compound XI WO2010015965, Compound XII WO2010015965, WO2009004071,WO2006038070, WO2008065666, Formula VII WO2010015965, Formula IVWO2010015965, dibenzoazepine, dibenzooxazepine, Formula I WO2009071631,N-{(1S)-1-[({(4R)-1-[(4-chlorophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-benzothiophen-2-carboxamide,N-{(1S)-1-[({(4R)-1-[(4-fluorophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-benzothiophen-2-carboxamide,N-{(1S)-1-[({(4R)-1-[(2-cyanophenyl)sulfonyl]-3-oxohexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-methyl-1H-indole-2-carboxamide,andN-{(1S)-1-[({(4R)-1-[(2-cyanophenyl)sulfonyl]hexahydro-1H-azepin-4-yl}amino)carbonyl]-3-methylbutyl}-1-methyl-1H-indole-2-carboxamide.

Suitable macromolecules include proteins and large peptides,polysaccharides and oligosaccharides, DNA and RNA nucleic acid moleculesand their analogs having therapeutic, prophylactic or diagnosticactivities. Proteins can include growth factors, hormones, cytokines(e.g., chemokines) and antibodies. As used herein, antibodies caninclude: all types of immunoglobulins, e.g., IgG, IgM, IgA, IgE, IgD,etc., from any source, e.g., human, rodent, rabbit, cow, sheep, pig,dog, other mammals, chicken, other avian, aquatic animal species etc.,monoclonal and polyclonal antibodies, single chain antibodies (includingIgNAR (single-chain antibodies derived from sharks)), chimericantibodies, bifunctional/bispecific antibodies, humanized antibodies,human antibodies, and complementary determining region (CDR)-graftedantibodies, that are specific for the target protein or fragmentsthereof, and also include antibody fragments, including Fab, Fab′,F(ab′)2, scFv, Fv, camelbodies, microantibodies, nanobodies, andsmall-modular immunopharmaceuticals (SMIPs). Nucleic acid moleculesinclude DNA, e.g., encoding genes or gene fragments, or RNA, includingmRNA, antisense molecules, such as antisense RNA, RNA molecules involvedin RNA interference (RNAi), such as microRNA (miRNA), small interferingRNA (siRNA) and small hairpin RNA (shRNA), ribozymes or other moleculescapable of inhibiting transcription and/or translation. Preferredmacromolecules have a molecular weight of at least 800 Da, at least 3000Da or at least 5000 Da.

In preferred embodiments, the respirable dry powder or respirable dryparticle comprises a therapeutic antibody. In certain preferredembodiments, the antibody is a monoclonal antibody. In certain preferredembodiments, the antibody is a single chain antibody, a chimericantibody, a bifunctional/bispecific antibody, a humanized antibody, or acombination thereof. In preferred embodiments, the antibody is selectedfrom the group consisting of: monoclonal antibodies, e.g., Abciximab(ReoPro®, chimeric), Adalimumab (Humira®, human), Alemtuzumab (Campath®,humanized), Basiliximab (Simulect®, chimeric), Belimumab (Benlysta®,human), Bevacizumab (Avastin®, humanized), Brentuximab vedotin(Adcetris®, chimeric), Canakinumab (Ilaris®, human), Cetuximab(Erbitux®, chimeric), Certolizumab pegol (Cimzia®, humanized),Daclizumab (Zenapax®, humanized), Denosumab (Prolia®, Xgeva®, human),Eculizumab (Soliris®, humanized), Efalizumab (Raptiva®, humanized),Gemtuzumab (Mylotarg®, humanized), Golimumab (Simponi®, human),Ibritumomab tiuxetan (Zevalin®, murin), Infliximab (Remicade®,chimeric), Ipilimumab (MDX-101) (Yervoy®, human), Muromonab-CD3(Orthoclone OKT3, murine), Natalizumab (Tysabri®, humanized), Ofatumumab(Arzerra®, human), Omalizumab (Xolair®, humanized), Palivizumab(Synagis®, humanized), Panitumumab (Vectibix®, human), Ranibizumab(Lucentis®, humanized), Rituximab (Rituxan®, Mabthera®, chimeric),Tocilizumab (or Atlizumab) (Actemra® and RoActemra®, humanized),Tositumomab (Bexxar®, murine), Trastuzumab (Herceptin®, humanized), andbispecific antibodies, e.g., catumaxomab (Removab®, rat-mouse hybridmonoclonal antibody).

Selected macromolecule active agents for systemic applications include,but are not limited to: Ventavis® (Iloprost), Calcitonin, Erythropoietin(EPO), Factor IX, Granulocyte Colony Stimulating Factor (G-CSF),Granulocyte Macrophage Colony, Stimulating Factor (GM-CSF), GrowthHormone, Insulin, TGF-beta, Interferon Alpha, Interferon Beta,Interferon Gamma, Luteinizing Hormone Releasing Hormone (LHRH), folliclestimulating hormone (FSH), Ciliary Neurotrophic Factor, Growth HormoneReleasing Factor (GRF), Insulin-Like Growth Factor, Insulinotropin,Interleukin-1 Receptor Antagonist, Interleukin-3, Interleukin-4,Interleukin-6, Macrophage Colony Stimulating Factor (M-CSF), ThymosinAlpha 1, IIb/IIIa Inhibitor, Alpha-1 Antitrypsin, Anti-RSV Antibody,palivizumab, motavizumab, and ALN-RSV, Cystic Fibrosis TransmembraneRegulator (CFTR) Gene, Deoxyribonuclase (DNase), Heparin,Bactericidal/Permeability Increasing Protein (BPI), Anti-Cytomegalovirus(CMV) Antibody, Interleukin-1 Receptor Antagonist, and the like,alpha-defensins (e.g., human neutrophil proteins (HNPs): HNP1, 2, 3, and4; human defensins 5 and 6 (HD5 and HD6)), beta-defensins (HBD1, 2, 3,and 4), or Θ-defensins/retrocyclins, GLP-1 analogs (liraglutide,exenatide, etc.), Domain antibodies (dAbs), Pramlintide acetate(Symlin), Leptin analogs, Synagis (palivizumab, MedImmune) andcisplatin. In certain preferred embodiments, the respirable dry powderor respirable dry particle comprises a macromolecule involved in intra-or inter-cellular signaling, such as a growth factor, a cytokine, achemokine or a hormone. In preferred embodiments, the respirable drypowder or respirable dry particle comprises a hormone. In certainpreferred embodiments, the hormone is insulin.

Selected therapeutics helpful for chronic maintenance of CF includeantibiotics/macrolide antibiotics, bronchodilators, inhaled LABAs, andagents to promote airway secretion clearance. Suitable examples ofantibiotics/macrolide antibiotics include tobramycin, azithromycin,ciprofloxacin, colistin, aztreonam and the like. Another exemplaryantibiotic/macrolide is levofloxacin. Suitable examples ofbronchodilators include inhaled short-acting beta₂ agonists such asalbuterol, and the like. Suitable examples of inhaled LABAs includesalmeterol, formoterol, and the like. Suitable examples of agents topromote airway secretion clearance include Pulmozyme (dornase alfa,Genentech) hypertonic saline, DNase, heparin, and the like. Selectedtherapeutics helpful for the prevention and/or treatment of CF includeVX-770 (Vertex Pharmaceuticals) and amiloride.

Selected therapeutics helpful for the treatment of idiopathic pulmonaryfibrosis include Metelimumab (CAT-192) (TGF-β1 mAb inhibitor, Genzyme),Aerovant™ (AER001, pitrakinra) (Dual IL-13, IL-4 protein antagonist,Aerovance), Aeroderm™ (PEGylated Aerovant, Aerovance), microRNA, RNAi,and the like.

In preferred embodiments, the respirable dry powder or respirable dryparticle comprises an antibiotic, such as a macrolide (e.g.,azithromycin, clarithromycin and erythromycin), a tetracycline (e.g.,doxycycline, tigecycline), a fluoroquinolone (e.g., gemifloxacin,levofloxacin, ciprofloxacin and mocifloxacin), a cephalosporin (e.g.,ceftriaxone, defotaxime, ceftazidime, cefepime), a penicillin (e.g.,amoxicillin, amoxicillin with clavulanate, ampicillin, piperacillin, andticarcillin) optionally with a β-lactamase inhibitor (e.g., sulbactam,tazobactam and clavulanic acid), such as ampicillin-sulbactam,piperacillin-tazobactam and ticarcillin with clavulanate, anaminoglycoside (e.g., amikacin, arbekacin, gentamicin, kanamycin,neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin,tobramycin, and apramycin), a penem or carbapenem (e.g., doripenem,ertapenem, imipenem and meropenem), a monobactam (e.g., aztreonam), anoxazolidinone (e.g., linezolid), vancomycin, glycopeptide antibiotics(e.g., telavancin), tuberculosis-mycobacterium antibiotics, tobramycin,azithromycin, ciprofloxacin, colistin, and the like. In a preferredembodiment, the respirable dry powder or respirable dry particlecomprises levofloxacin. In another preferred embodiment, the respirabledry powder or respirable dry particle comprises aztreonam (i.e.,Cayston®) or a pharmaceutically acceptable salt thereof. In a furtherpreferred embodiment, the respirable dry powder or respirable dryparticle does not comprise tobramycin. In another embodiment, therespirable dry powder or respirable dry particle does not compriselevofloxacin. In another embodiment, the respirable dry powder orrespirable dry particle does not comprise Cayston®.

In preferred embodiments, the respirable dry powder or respirable dryparticle comprises a LABA, such as salmeterol, formoterol and isomers(e.g., arformoterol), clenbuterol, tulobuterol, vilanterol (Revolair™),indacaterol, carmoterol, isoproterenol, procaterol, bambuterol,milveterol, and the like. In a further preferred embodiment, therespirable dry powder or respirable dry particle comprises formoterol.In a further preferred embodiment, the respirable dry powder orrespirable dry particle comprises salmeterol. When the dry powders areintended for treatment of CF, preferred additional therapeutic agentsare short-acting beta agonists (e.g., albuterol), antibiotics (e.g.,levofloxacin), recombinant human deoxyribonuclease I (e.g., dornasealfa, also known as DNase), sodium channel blockers (e.g., amiloride),and combinations thereof.

In preferred embodiments, the respirable dry powder or respirable dryparticle comprises a LAMA, such as tiotroprium, glycopyrrolate,aclidinium, ipratropium and the like. In a further preferred embodiment,the respirable dry powder or respirable dry particle comprisestiotropium.

In preferred embodiments, the respirable dry powder or respirable dryparticle comprises a corticosteroid, such as budesonide, fluticasone,flunisolide, triamcinolone, beclomethasone, mometasone, ciclesonide,dexamethasone, and the like. In a further preferred embodiment, therespirable dry powder or respirable dry particle comprises fluticasone.

Preferred additional therapeutic agents are LABAs (e.g., formoterol,salmeterol), short-acting beta agonists (e.g., albuterol),corticosteroids (e.g., fluticasone), LAMAs (e.g., tiotropium),antibiotics (e.g., levofloxacin, tobramycin), and combinations thereof.When the dry powders are intended for treatment of CF, preferredadditional therapeutic agents are short-acting beta agonists (e.g.,albuterol), antibiotics (e.g., levofloxacin), recombinant humandeoxyribonuclease I (e.g., dornase alfa, also known as DNAse), sodiumchannel blockers (e.g., amiloride), and combinations thereof.

In preferred embodiments, the respirable dry powder or respirable dryparticle comprises a combination of two or more of the following; aLABA, a LAMA, and a corticosteroid. In a further preferred embodiment,the respirable dry powder or respirable dry particle comprisesfluticasone and salmeterol. In a further preferred embodiment, therespirable dry powder or respirable dry particle comprises fluticasone,salmeterol, and tiotropium.

When an additional therapeutic agent is administered to a patient with adry powder or dry particles disclosed herein, the agent and the drypowder or dry particles are administered to provide overlap of thetherapeutic effect of the additional therapeutic agent with theadministration of the dry powder or dry particles. For example, a LABAsuch as formoterol, or a short-acting beta agonist such as albuterol canbe administered to the patient before a dry powder or dry particle, asdescribed herein, is administered.

Dry Powder and Dry Particle Properties

The dry particles of the invention are small and dispersible, andpreferably dense (e.g., active agent dense and/or mass dense).Generally, the dry particles of the invention have a VMGD as measured byHELOS/RODOS at 1.0 bar of about 10 μm or less (e.g., about 0.1 μm toabout 10 μm). Preferably, the dry particles of the invention have anVMGD of about 9 μm or less (e.g., about 0.1 μm to about 9 μm), about 8μm or less (e.g., about 0.1 μm to about 8 μm), about 7 μm or less (e.g.,about 0.1 μm to about 7 μm), about 6 μm or less (e.g., about 0.1 μm toabout 6 μm), about 5 μm or less (e.g., less than 5 μm, about 0.1 μm toabout 5 μm), about 4 μm or less (e.g., 0.1 μm to about 4 μm), about 3 μmor less (e.g., 0.1 μm to about 3 μm), about 2 μm or less (e.g., 0.1 μmto about 2 μm), about 1 μm or less (e.g., 0.1 μm to about 1 μm), about 1μm to about 6 μm, about 1 μm to about 5 μm, about 1 μm to about 4 μm,about 1 μm to about 3 μm, or about 1 μm to about 2 μm as measured byHELOS/RODOS at 1.0 bar.

In another aspect, the dry particles of the invention are large andpreferably dense (e.g., active agent dense), and are dispersible.Generally, the dry particles of the invention have a VMGD as measured byHELOS/RODOS at 1.0 bar of about 30 μm or less (e.g., about 5 μm to about30 μm). Preferably, the dry particles of the invention have an VMGD ofabout 25 μm or less (e.g., about 5 μm to about 25 μm), about 20 μm orless (e.g., about 5 μm to about 20 μm), about 15 μm or less (e.g., about5 μm to about 15 μm), about 12 μm or less (e.g., about 5 μm to about 12μm), about 10 μm or less (e.g., about 5 μm to about 10 μm), or about 8μm or less (e.g., 6 μm to about 8 μm) as measured by HELOS/RODOS at 1.0bar.

The respirable dry powders of the invention can have poor flowproperties, such as bulk flow properties, for example as assessed byHausner Ratio, as described herein. Yet, surprisingly, the powders arehighly dispersible. This is surprising because flow properties anddispersibility are both known to be negatively affected by particleagglomeration or aggregation. Thus, it was unexpected that particlesthat have poor flow characteristics, such as bulk flow characteristics,would be highly dispersible.

Generally, the respirable dry powders can have a Hausner Ratio that isgreater than 1.5, and can be 1.6 or higher, 1.7 or higher, 1.8 orhigher, 1.9 or higher, 2 or higher, 2.1 or higher, 2.2 or higher, 2.3 orhigher, 2.4 or higher, 2.5 or higher, 2.6 or higher or 2.7 or higher,between 2.2 and 2.9, between 2.2 and 2.8, between 2.2 and 2.7, between2.2 and 2.6, between 2.2 and 2.5, between 2.3 and 2.5, between 2.6 and2.8, about 2.7, or about 2.4.

The dry particles of the invention are dispersible, and have 1 bar/4 barand/or 0.5 bar/4 bar of about 2.2 or less (e.g., about 1.0 to about 2.2)or about 2.0 or less (e.g., about 1.0 to about 2.0). Preferably, the dryparticles of the invention have 1 bar/4 bar and/or 0.5 bar/4 bar ofabout 1.9 or less (e.g., about 1.0 to about 1.9), about 1.8 or less(e.g., about 1.0 to about 1.8), about 1.7 or less (e.g., about 1.0 toabout 1.7), about 1.6 or less (e.g., about 1.0 to about 1.6), about 1.5or less (e.g., about 1.0 to about 1.5), about 1.4 or less (e.g., about1.0 to about 1.4), about 1.3 or less (e.g., less than 1.3, about 1.0 toabout 1.3), about 1.2 or less (e.g., 1.0 to about 1.2), about 1.1 orless (e.g., 1.0 to about 1.1 μm) or the dry particles of the inventionhave 1 bar/4 bar and/or 0.5 bar/4 bar of about 1.0.

Alternatively or in addition, the respirable dry particles of theinvention can have an MMAD of about 10 microns or less, such as an MMADof about 0.5 micron to about 10 microns. Preferably, the dry particlesof the invention have an MMAD of about 5 microns or less (e.g., about0.5 micron to about 5 microns, preferably about 1 micron to about 5microns), about 4 microns or less (e.g., about 1 micron to about 4microns), about 3.8 microns or less (e.g., about 1 micron to about 3.8microns), about 3.5 microns or less (e.g., about 1 micron to about 3.5microns), about 3.2 microns or less (e.g., about 1 micron to about 3.2microns), about 3 microns or less (e.g., about 1 micron to about 3.0microns), about 2.8 microns or less (e.g., about 1 micron to about 2.8microns), about 2.2 microns or less (e.g., about 1 micron to about 2.2microns), about 2.0 microns or less (e.g., about 1 micron to about 2.0microns) or about 1.8 microns or less (e.g., about 1 micron to about 1.8microns).

Alternatively or in addition, the respirable dry powders and dryparticles of the invention can have an FPF of less than about 5.6microns of the total dose (FPF<5.6 μm) of at least about 20%, at leastabout 30%, at least about 40%, preferably at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,or at least about 70%.

Alternatively or in addition, the dry powders and dry particles of theinvention have a FPF of less than 5.0 microns (FPF_TD<5.0 μm) of atleast about 20%, at least about 30%, at least about 45%, preferably atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 65% or at least about 70%. Alternatively or inaddition, the dry powders and dry particles of the invention have a FPFof less than 5.0 microns of the emitted dose (FPF_ED<5.0 μm) of at leastabout 45%, preferably at least about 50%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,or at least about 85%. Alternatively or in addition, the dry powders anddry particles of the invention can have an FPF of less than about 3.4microns (FPF<3.4 μm) of at least about 20%, preferably at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, or at least about 55%.

Alternatively or in addition, the respirable dry powders and dryparticles of the invention have a tap density of about 0.1 g/cm³ toabout 1.0 g/cm³. For example, the small and dispersible dry particleshave a tap density of about 0.1 g/cm³ to about 0.9 g/cm³, about 0.2g/cm³ to about 0.9 g/cm³, about 0.2 g/cm³ to about 0.9 g/cm³, about 0.3g/cm³ to about 0.9 g/cm³, about 0.4 g/cm³ to about 0.9 g/cm³, about 0.5g/cm³ to about 0.9 g/cm³, or about 0.5 g/cm³ to about 0.8 g/cm³, greaterthan about 0.4 g/cc, greater than about 0.5 g/cc, greater than about 0.6g/cc, greater than about 0.7 g/cc, about 0.1 g/cm³ to about 0.8 g/cm³,about 0.1 g/cm³ to about 0.7 g/cm³, about 0.1 g/cm³ to about 0.6 g/cm³,about 0.1 g/cm³ to about 0.5 g/cm³, about 0.1 g/cm³ to about 0.4 g/cm³,about 0.1 g/cm³ to about 0.3 g/cm³, less than 0.3 g/cm³. In a preferredembodiment, tap density is greater than about 0.4 g/cc. In anotherpreferred embodiment, tap density is greater than about 0.5 g/cc.Alternatively, tap density is less than about 0.4 g/cc.

Alternatively or in addition, the respirable dry powders and dryparticles of the invention can have a water or solvent content of lessthan about 15% by weight of the respirable dry particle. For example,the respirable dry particles of the invention can have a water orsolvent content of less than about 15% by weight, less than about 13% byweight, less than about 11.5% by weight, less than about 10% by weight,less than about 9% by weight, less than about 8% by weight, less thanabout 7% by weight, less than about 6% by weight, less than about 5% byweight, less than about 4% by weight, less than about 3% by weight, lessthan about 2% by weight, less than about 1% by weight or be anhydrous.The respirable dry particles of the invention can have a water orsolvent content of less than about 6% and greater than about 1%, lessthan about 5.5% and greater than about 1.5%, less than about 5% andgreater than about 2%, about 2%, about 2.5%, about 3%, about 3.5%, about4%, about 4.5% about 5%.

The respirable dry particles can be characterized by the crystalline andamorphous content of the particles. The respirable dry particles cancomprise a mixture of amorphous and crystalline content, in which thedivalent metal cation salt, e.g., calcium salt and/or magnesium salt, issubstantially in the amorphous phase. If a monovalent salt is present,e.g., a sodium salt, it can be substantially in the crystalline phase.As described herein, the respirable dry particles can further comprisean excipient, such as leucine, maltodextrin or mannitol, and/or apharmaceutically active agent. The excipient and pharmaceutically activeagent can independently be crystalline or amorphous or present in acombination of these forms. In some embodiments, the excipient isamorphous or predominately amorphous.

This provides several advantages. For example, the crystalline phase(e.g., crystalline sodium chloride) can contribute to the stability ofthe dry particle in the dry state and to the dispersibilitycharacteristics, whereas the amorphous phase (e.g., amorphous activeagent and/or excipient) can facilitate rapid water uptake anddissolution of the particle upon deposition in the respiratory tract. Itis particularly advantageous when salts with relatively high aqueoussolubilities (such as sodium chloride) that are present in the dryparticles are in a crystalline state and when salts with relatively lowaqueous solubilities (such as calcium citrate) are present in the dryparticles in an amorphous state.

The amorphous phase can be characterized by a high glass transitiontemperature (T_(g)), such as a T_(g) of at least 90° C., at least 100°C., at least 105° C., at least 110° C., at least 115° C., 120° C., atleast 125° C., at least 130° C., at least 135° C., at least 140° C.,between 120° C. and 200° C., between 125° C. and 200° C., between 130°C. and 200° C., between 120° C. and 190° C., between 125° C. and 190°C., between 130° C. and 190° C., between 120° C. and 180° C., between125° C. and 180° C., or between 130° C. and 180° C. Alternatively, theamorphous phase can be characterized by a high T_(g) such as at least80° C. or at least 90° C.

In some embodiments, the respirable dry particles contain an excipientand or active agent rich amorphous phase and a monovalent salt (sodiumsalt, potassium salt) crystalline phase and the ratio of amorphous phaseto crystalline phase (w:w) is about 5:95 to about 95:5, about 5:95 toabout 10:90, about 10:90 to about 20:80, about 20:80 to about 30:70,about 30:70 to about 40:60, about 40:60 to about 50:50; about 50:50 toabout 60:40, about 60:40 to about 70:30, about 70:30 to about 80:20, orabout 90:10 to about 95:5. In other embodiments, the respirable dryparticles contain an amorphous phase and a monovalent salt crystallinephase and the ratio of amorphous phase to particle by weight (w:w) isabout 5:95 to about 95:5, about 5:95 to about 10:90, about 10:90 toabout 20:80, about 20:80 to about 30:70, about 30:70 to about 40:60,about 40:60 to about 50:50; about 50:50 to about 60:40, about 60:40 toabout 70:30, about 70:30 to about 80:20, or about 90:10 to about 95:5.In other embodiments, the respirable dry particles contain an amorphousphase and a monovalent salt crystalline phase and the ratio ofcrystalline phase to particle by weight (w:w) is about 5:95 to about95:5, about 5:95 to about 10:90, about 10:90 to about 20:80, about 20:80to about 30:70, about 30:70 to about 40:60, about 40:60 to about 50:50;about 50:50 to about 60:40, about 60:40 to about 70:30, about 70:30 toabout 80:20, or about 90:10 to about 95:5.

In addition to any of the features and properties described herein, inany combination, the respirable dry particles can have a heat ofsolution that is not highly exothermic. Preferably, the heat of solutionis determined using the ionic liquid of a simulated lung fluid (e.g., asdescribed in Moss, O.R. 1979. Simulants of lung interstitial fluid.Health Phys. 36, 447-448; or in Sun, G. 2001. Oxidative interactions ofsynthetic lung epithelial lining fluid with metal-containing particulatematter. Am J Physiol Lung Cell Mol. Physiol. 281, L807-L815) at pH 7.4and 37° C. in an isothermal calorimeter. For example, the respirable dryparticles can have a heat of solution that is less exothermic than theheat of solution of calcium chloride dihydrate, e.g., have a heat ofsolution that is greater than about −10 kcal/mol, greater than about −9kcal/mol, greater than about −8 kcal/mol, greater than about −7kcal/mol, greater than about −6 kcal/mol, greater than about −5kcal/mol, greater than about −4 kcal/mol, greater than about −3kcal/mol, greater than about −2 kcal/mol, greater than about −1 kcal/molor about −10 kcal/mol to about 10 kcal/mol. Alternatively, a preferredΔH is between about −9 kcal/mol and about 9 kcal/mol, between about −8kcal/mol and about 8 kcal/mol, between about −7 kcal/mol and about 7kcal/mol, between about −6 kcal/mol and about 6 kcal/mol, between about−5 kcal/mol and about 5 kcal/mol, between about −4 kcal/mol and about 4kcal/mol, between about −3 kcal/mol and about 3 kcal/mol, between about−2 kcal/mol and about 2 kcal/mol, between about −1 kcal/mol and about 1kcal/mol, or about 0 kcal/mol.

The respirable dry powders and dry particles are characterized by a highemitted dose (e.g., CEPM of at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%) from a dry powderinhaler when a total inhalation energy of less than about 2 Joules orless than about 1 Joule, or less than about 0.8 Joule, or less thanabout 0.5 Joule, or less than about 0.3 Joule is applied to the drypowder inhaler. For example, an emitted dose of at at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% CEPM of respirabledry powder contained in a unit dose container, containing about 50 mg ofthe appropriate formulation, in a dry powder inhaler can be achievedwhen a total inhalation energy of less than about 1 Joule (e.g., lessthan about 0.8 Joule, less than about 0.5 Joule, less than about 0.3Joule) is applied to the dry powder inhaler. An emitted dose of at leastabout 70% CEPM of Formulation I or Formulation II contained in a unitdose container, containing about 50 mg of the appropriate formulation,in a dry powder inhaler can be achieved when a total inhalation energyof less than about 0.28 Joule is applied to the dry powder inhaler. Thedry powder can fill the unit dose container, or the unit dose containercan be at least 40% full, at least 50% full, at least 60% full, at least70% full, at least 80% full, or at least 90% full. The unit dosecontainer can be a capsule (e.g., size 000, 00, 0E, 0, 1, 2, 3, and 4,with respective volumetric capacities of 1.37 ml, 950 μl, 770 μl, 680μl, 480 μl, 360 μl, 270 μl, and 200 μl).

Healthy adult populations are predicted to be able to achieve inhalationenergies ranging from 2.9 Joules for comfortable inhalations to 22Joules for maximum inhalations by using values of peak inspiratory flowrate (PIFR) measured by Clarke et al. (Journal of Aerosol Med, 6(2), p.99-110, 1993) for the flow rate Q from two inhaler resistances of 0.02and 0.055 kPa1/2/LPM, with a inhalation volume of 2 L based on both FDAguidance documents for dry powder inhalers and on the work of Tiddens etal. (Journal of Aerosol Med, 19, (4), p. 456-465, 2006) who found adultsaveraging 2.2 L inhaled volume through a variety of DPIs.

Mild, moderate and severe adult COPD patients are predicted to be ableto achieve maximum inhalation energies of 5.1 to 21 Joules, 5.2 to 19Joules, and 2.3 to 18 Joules respectively. This is again based on usingmeasured PIFR values for the flow rate Q in the equation for inhalationenergy. The PIFR achievable for each group is a function of the inhalerresistance that is being inhaled through. The work of Broeders et al.(Eur Respir J, 18, p. 780-'783, 2001) was used to predict maximum andminimum achievable PIFR through 2 dry powder inhalers of resistances0.021 and 0.032 kPa1/2/LPM for each.

Similarly, adult asthmatic patients are predicted to be able to achievemaximum inhalation energies of 7.4 to 21 Joules based on the sameassumptions as the COPD population and PIFR data from Broeders et al.

Healthy adults and children, COPD patients, asthmatic patients ages 5and above, and CF patients, for example, are capable of providingsufficient inhalation energy to empty and disperse the dry powderformulations of the invention.

An advantage of aspects of the invention is the production of powdersthat disperse well across a wide range of flow rates and are relativelyflow rate independent. In certain aspects, the dry particles and powdersof the invention enable the use of a simple, passive DPI for a widepatient population.

Preferrably, the respirable dry particles have a 1 bar/4 bar or 0.5bar/4 bar of 2 or less, as described herein. For example, a 1 bar/4 baror 0.5 bar/4 bar of 1.9 or less, 1.8 or less, 1.7 or less, 1.6 or less,1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or less or about1.0. Alternatively or in addition, the respirable dry particles have anMMAD of about 5 microns or less. Alternatively or in addition, therespirable dry particles can have a VMGD between about 0.5 microns andabout 5 microns, or a VMGD between about 5 microns and about 20 microns.Alternatively or in addition, the respirable dry particles can have aheat of solution that not is greater than about −10 kcal/mol (e.g.,between −10 kcal/mol and 10 kcal/mol or between −7 kcal/mol and 7kcal/mol).

In preferred aspects, the respirable dry powder comprises respirable dryparticles that characterized by:

1. VMGD at 1 bar as measured using a HELOS/RODOS system between 0.5microns and 10 microns, preferably between 1 microns and 7 microns,between 1 microns and 5 microns, or between 1 microns and 3 microns;

2. 1 bar/4 bar of 1.6 or less, preferably less than 1.5, less than 1.4,less than 1.3, less than 1.2 or less than 1.1; and

3. tap density of about 0.4 g/cm³ to about 1.2 g/cm³, 0.5 g/cm³ to about1.0 g/cm³, preferably between about 0.6 g/cm³ and about 0.9 g/cm³.

In other preferred aspects, the respirable dry powder comprisesrespirable dry particles that characterized by:

1. VMGD at 1 bar as measured using a HELOS/RODOS system between 0.5microns and 10 microns, preferably between 1 microns and 7 microns,between 1 microns and 5 microns, or between 1 microns and 3 microns;

2. 1 bar/4 bar of 1.6 or less, preferably less than 1.5, less than 1.4,less than 1.3, less than 1.2 or less than 1.1; and

3. MMAD between 0.5 and 6.0, between 1.0 and 5.0 or between 1.0 and 3.0.

In other preferred aspects, the respirable dry powder comprisesrespirable dry particles that are characterized by:

1. VMGD at 1 bar as measured using a HELOS/RODOS system between 0.5microns and 10 microns, preferably between 1 microns and 7 microns,between 1 microns and 5 microns, or between 1 microns and 3 microns;

2. 1 bar/4 bar of 1.6 or less, preferably less than 1.5, less than 1.4,less than 1.3, less than 1.2 or less than 1.1; and

3. FPF_TD<5.0 μm of at least 30%, at least 40%, at least 50% or at least60%.

In a further preferred aspect, the respirable dry powder comprisesrespirable dry particles that characterized by:

1. VMGD at 1 bar as measured using a HELOS/RODOS system between 0.5microns and 10 microns, preferably between 1 microns and 7 microns,between 1 microns and 5 microns, or between 1 microns and 3 microns;

2. 1 bar/4 bar of 1.6 or less, preferably less than 1.5, less than 1.4,less than 1.3, less than 1.2 or less than 1.1; and

3. Hausner Ratio greater than 1.5, greater than 1.8, or greater than2.1.

In other preferred aspects, the respirable dry powder comprisesrespirable dry particles that are characterized by:

1. tap density of about 0.4 g/cm³ to about 1.2 g/cm³, 0.5 g/cm³ to about1.0 g/cm³, preferably between about 0.6 g/cm³ and about 0.9 g/cm³.

2. FPF_TD<5.0 μm of at least 30%, at least 40%, at least 50% or at least60%.

3. Hausner Ratio greater than 1.5, greater than 1.8, or greater than2.1.

The respirable dry particles and dry powders described herein aresuitable for inhalation therapies. The respirable dry particles may befabricated with the appropriate material, surface roughness, diameter,magnetic properties and tap density for localized delivery to selectedregions of the respiratory system such as the deep lung or upper orcentral airways. For example, higher density or larger respirable dryparticles may be used for upper airway delivery, or a mixture of varyingsize respirable dry particles in a sample, provided with the same or adifferent formulation, may be administered to target different regionsof the lung in one administration. In one aspect, the magneticproperties refer to particle to particle charge properties.

Because the respirable dry powders and respirable dry particlesdescribed herein contain salts, they may be hygroscopic. Accordingly itis desirable to store or maintain the respirable dry powders andrespirable dry particles under conditions to prevent hydration of thepowders. For example, if it is desirable to prevent hydration, therelative humidity of the storage environment should be less than 75%,less than 60%, less than 50%, less than 40%, less than 30%, less than25%, less than 20%, less than 15%, less than 10%, or less than 5%humidity. The respirable dry powders and respirable dry particles can bepackaged (e.g., in sealed capsules, blisters, vials) under theseconditions.

In preferred embodiments, the respirable dry powders or respirable dryparticles of the invention possess aerosol characteristics that permiteffective delivery of the respirable dry particles to the respiratorysystem without the use of propellants.

The dry particles of the invention can be blended with an activeingredient or co-formulated with an active ingredient to maintaincharacteristic high dispersibility of the dry particles and dry powdersof the invention.

Methods for Preparing Dry Powders and Dry Particles

The respirable dry particles and dry powders can be prepared using anysuitable method. Many suitable methods for preparing respirable drypowders and particles are conventional in the art, and include singleand double emulsion solvent evaporation, spray drying, milling (e.g.,jet milling), blending, solvent extraction, solvent evaporation, phaseseparation, simple and complex coacervation, interfacial polymerization,suitable methods that involve the use of supercritical carbon dioxide(CO₂), sonocrystallization, nanoparticle aggregate formation and othersuitable methods. Respirable dry particles can be made using methods formaking microspheres or microcapsules known in the art. These methods canbe employed under conditions that result in the formation of respirabledry particles with desired aerodynamic properties (e.g., aerodynamicdiameter and geometric diameter). If desired, respirable dry particleswith desired properties, such as size and density, can be selected usingsuitable methods, such as sieving.

The respirable dry particles are preferably spray dried. Suitablespray-drying techniques are described, for example, by K. Masters in“Spray Drying Handbook”, John Wiley & Sons, New York (1984). Generally,during spray-drying, heat from a hot gas such as heated air or nitrogenis used to evaporate a solvent from droplets formed by atomizing acontinuous liquid feed. If desired, the spray drying or otherinstruments, e.g., jet milling instrument, used to prepare the dryparticles can include an inline geometric particle sizer that determinesa geometric diameter of the respirable dry particles as they are beingproduced, and/or an inline aerodynamic particle sizer that determinesthe aerodynamic diameter of the respirable dry particles as they arebeing produced.

For spray drying, solutions, emulsions or suspensions that contain thecomponents of the dry particles to be produced in a suitable solvent(e.g., aqueous solvent, organic solvent, aqueous-organic mixture oremulsion) are distributed to a drying vessel via an atomization device.For example, a nozzle or a rotary atomizer may be used to distribute thesolution or suspension to the drying vessel. For example, a rotaryatomizer having a 4- or 24-vaned wheel may be used. Examples of suitablespray dryers that can be outfitted with either a rotary atomizer or anozzle, include, Mobile Minor Spray Dryer or the Model PSD-1, bothmanufactured by GEA Group (Niro, Inc.; Denmark). Actual spray dryingconditions will vary depending, in part, on the composition of the spraydrying solution or suspension and material flow rates. The person ofordinary skill will be able to determine appropriate conditions based onthe compositions of the solution, emulsion or suspension to be spraydried, the desired particle properties and other factors. In general,the inlet temperature to the spray dryer is about 90° C. to about 300°C., and preferably is about 220° C. to about 285° C. The spray dryeroutlet temperature will vary depending upon such factors as the feedtemperature and the properties of the materials being dried. Generally,the outlet temperature is about 50° C. to about 150° C., preferablyabout 90° C. to about 120° C., or about 98° C. to about 108° C.

In another aspect, the inlet temperature to the spray dryer is about 90°C. to about 300° C., and preferably is about 150° C. to about 220° C.The spray dryer outlet temperature will vary depending upon such factorsas the feed temperature and the properties of the materials being dried.Generally, the outlet temperature is about 50° C. to about 150° C.,preferably about 50° C. to about 90° C.

If desired, the respirable dry particles that are produced can befractionated by volumetric size, for example, using a sieve, orfractioned by aerodynamic size, for example, using a cyclone, and/orfurther separated according to density using techniques known to thoseof skill in the art.

To prepare the respirable dry particles of the invention, generally, asolution, emulsion or suspension that contains the desired components ofthe dry powder (i.e., a feed stock) is prepared and spray dried undersuitable conditions. Preferably, the dissolved or suspended solidsconcentration in the feed stock is at least about 1 g/L, at least about2 g/L, at least about 5 g/L, at least about 10 g/L, at least about 15g/L, at least about 20 g/L, at least about 30 g/L, at least about 40g/L, at least about 50 g/L, at least about 60 g/L, at least about 70g/L, at least about 80 g/L, at least about 90 g/L, or at least about 100g/L. The feed stock can be provided by preparing a single solution orsuspension by dissolving or suspending suitable components (e.g., salts,excipients, other active ingredients) in a suitable solvent. Thesolvent, emulsion or suspension can be prepared using any suitablemethods, such as bulk mixing of dry and/or liquid components or staticmixing of liquid components to form a combination. For example, ahydrophilic component (e.g., an aqueous solution) and a hydrophobiccomponent (e.g., an organic solution) can be combined using a staticmixer to form a combination. The combination can then be atomized toproduce droplets, which are dried to form respirable dry particles.Preferably, the atomizing step is performed immediately after thecomponents are combined in the static mixer.

The feed stock, or components of the feed stock, can be prepared usingany suitable solvent, such as an organic solvent, an aqueous solvent ormixtures thereof. Suitable organic solvents that can be employed includebut are not limited to alcohols such as, for example, ethanol, methanol,propanol, isopropanol, butanols, and others. Other organic solventsinclude but are not limited to perfluorocarbons, dichloromethane,chloroform, ether, ethyl acetate, methyl tert-butyl ether and others.Co-solvents that can be employed include an aqueous solvent and anorganic solvent, such as, but not limited to, the organic solvents asdescribed above. Aqueous solvents include water and buffered solutions.

The feed stock or components of the feed stock can have any desired pH,viscosity or other properties. If desired, a pH buffer can be added tothe solvent or co-solvent or to the formed mixture. Generally, the pH ofthe mixture ranges from about 3 to about 8.

Respirable dry particles and dry powders can be fabricated and thenseparated, for example, by filtration or centrifugation by means of acyclone, to provide a particle sample with a preselected sizedistribution. For example, greater than about 30%, greater than about40%, greater than about 50%, greater than about 60%, greater than about70%, greater than about 80%, or greater than about 90% of the respirabledry particles in a sample can have a diameter within a selected range.The selected range within which a certain percentage of the respirabledry particles fall can be, for example, any of the size ranges describedherein, such as between about 0.1 to about 3 microns VMGD.

The invention also relates to respirable dry powders or respirable dryparticles produced by preparing a feedstock solution, emulsion orsuspension and spray drying the feedstock according to the methodsdescribed herein. The feedstock can be prepared using (a) e.g., acalcium salt, such as calcium lactate or calcium chloride, or amagnesium salt that provide divalent metal cation in an amount of lessthan 3% by weight and (b) a sodium salt, such as sodium citrate, sodiumchloride or sodium sulfate, in an amount of at least about 1% to 99.9%by weight of the resulting dry particle. If desired, an excipient, suchas leucine can be added to the feedstock in an amount of about 0% to 99%by weight of the resulting dry particle, and optionally apharmaceutically active agent in an amount of about 0.001% to 99% byweight of the resulting dry particle and one or more suitable solventsfor dissolution of the solutes and formulations of the feedstock.

Any suitable method can be used for mixing the solutes and solvents toprepare feedstocks (e.g., static mixing, bulk mixing). If desired,additional components that cause or facilitate the mixing can beincluded in the feedstock. For example, carbon dioxide produces fizzingor effervescence and thus can serve to promote physical mixing of thesolute and solvents. Various salts of carbonate or bicarbonate canpromote the same effect that carbon dioxide produces and, therefore, canbe used in preparation of the feedstocks of the invention.

In an embodiment, the respirable dry powders or respirable dry particlesof the invention can be produced through an ion exchange reaction. Incertain embodiments of the invention, two saturated or sub-saturatedsolutions are fed into a static mixer in order to obtain a saturated orsupersaturated solution post-static mixing. Preferably, the post-mixedsolution is supersaturated. The two solutions may be aqueous or organic,but are preferably substantially aqueous. The post-static mixingsolution is then fed into the atomizing unit of a spray dryer. In apreferable embodiment, the post-static mixing solution is immediatelyfed into the atomizer unit. Some examples of an atomizer unit include atwo-fluid nozzle, a rotary atomizer, or a pressure nozzle. Preferably,the atomizer unit is a two-fluid nozzle. In one embodiment, thetwo-fluid nozzle is an internally mixing nozzle, meaning that the gasimpinges on the liquid feed before exiting to most outward orifice. Inanother embodiment, the two-fluid nozzle is an externally mixing nozzle,meaning that the gas impinges on the liquid feed after exiting the mostoutward orifice.

The diameter of the respirable dry particles, for example, their VMGD,can be measured using an electrical zone sensing instrument such as aMultisizer He, (Coulter Electronic, Luton, Beds, England), or a laserdiffraction instrument such as a HELOS system (Sympatec, Princeton,N.J.) or a Mastersizer system (Malvern, Worcestershire, UK). Otherinstruments for measuring particle geometric diameter are well known inthe art. The diameter of respirable dry particles in a sample will rangedepending upon factors such as particle composition and methods ofsynthesis. The distribution of size of respirable dry particles in asample can be selected to permit optimal deposition within targetedsites within the respiratory system.

Experimentally, aerodynamic diameter can be determined using time offlight (TOF) measurements. For example, an instrument such as theAerosol Particle Sizer (APS) Spectrometer (TSI Inc., Shoreview, MNcan beused to measure aerodynamic diameter. The APS measures the time takenfor individual respirable dry particles to pass between two fixed laserbeams.

Aerodynamic diameter also can be experimentally determined directlyusing conventional gravitational settling methods, in which the timerequired for a sample of respirable dry particles to settle a certaindistance is measured. Indirect methods for measuring the mass medianaerodynamic diameter include the Andersen Cascade Impactor and themulti-stage liquid impinger (MSLI) methods. The methods and instrumentsfor measuring particle aerodynamic diameter are well known in the art.

Tap density is a measure of the envelope mass density characterizing aparticle. The envelope mass density of a particle of a statisticallyisotropic shape is defined as the mass of the particle divided by theminimum sphere envelope volume within which it can be enclosed. Featureswhich can contribute to low tap density include irregular surfacetexture, high particle cohesiveness and porous structure. Tap densitycan be measured by using instruments known to those skilled in the artsuch as the Dual Platform Microprocessor Controlled Tap Density Tester(Vankel, N.C.), a GeoPyc™ instrument (Micrometrics Instrument Corp.,Norcross, Ga.), or SOTAX Tap Density Tester model TD2 (SOTAX Corp.,Horsham, Pa.). Tap density can be determined using the method of USPBulk Density and Tapped Density, United States Pharmacopeia convention,Rockville, Md., 10^(th) Supplement, 4950-4951, 1999.

Fine particle fraction can be used as one way to characterize theaerosol performance of a dispersed powder. Fine particle fractiondescribes the size distribution of airborne respirable dry particles.Gravimetric analysis, using a Cascade impactor, is one method ofmeasuring the size distribution, or fine particle fraction, of airbornerespirable dry particles. The Andersen Cascade Impactor (ACI) is aneight-stage impactor that can separate aerosols into nine distinctfractions based on aerodynamic size. The size cutoffs of each stage aredependent upon the flow rate at which the ACI is operated. The ACI ismade up of multiple stages consisting of a series of nozzles (i.e., ajet plate) and an impaction surface (i.e., an impaction disc). At eachstage an aerosol stream passes through the nozzles and impinges upon thesurface. Respirable dry particles in the aerosol stream with a largeenough inertia will impact upon the plate. Smaller respirable dryparticles that do not have enough inertia to impact on the plate willremain in the aerosol stream and be carried to the next stage. Eachsuccessive stage of the ACI has a higher aerosol velocity in the nozzlesso that smaller respirable dry particles can be collected at eachsuccessive stage.

If desired, a two-stage collapsed ACI can also be used to measure fineparticle fraction. The two-stage collapsed ACI consists of only the toptwo stages 0 and 2 of the eight-stage ACI, as well as the finalcollection filter, and allows for the collection of two separate powderfractions. Specifically, a two-stage collapsed ACI is calibrated so thatthe fraction of powder that is collected on stage two is composed ofrespirable dry particles that have an aerodynamic diameter of less than5.6 microns and greater than 3.4 microns. The fraction of powder passingstage two and depositing on the final collection filter is thus composedof respirable dry particles having an aerodynamic diameter of less than3.4 microns. The airflow at such a calibration is approximately 60L/min. The FPF(<5.6) has been demonstrated to correlate to the fractionof the powder that is able to reach the lungs of the patient, while theFPF(<3.4) has been demonstrated to correlate to the fraction of thepowder that reaches the deep lung of a patient. These correlationsprovide a quantitative indicator that can be used for particleoptimization.

The FPF(<5.6) has been demonstrated to correlate to the fraction of thepowder that is able to make it into the lung of the patient, while theFPF(<3.4) has been demonstrated to correlate to the fraction of thepowder that reaches the deep lung of a patient. These correlationsprovide a quantitative indicator that can be used for particleoptimization.

An ACI can be used to approximate the emitted dose, which herein iscalled gravimetric recovered dose and analytical recovered dose.“Gravimetric recovered dose” is defined as the ratio of the powderweighed on all stage filters of the ACI to the nominal dose. “Analyticalrecovered dose” is defined as the ratio of the powder recovered fromrinsing all stages, all stage filters, and the induction port of the ACIto the nominal dose. The FPF_TD(<5.0) is the ratio of the interpolatedamount of powder depositing below 5.0 μm on the ACI to the nominal dose.The FPF_RD(<5.0) is the ratio of the interpolated amount of powderdepositing below 5.0 μam on the ACI to either the gravimetric recovereddose or the analytical recovered dose.

Another way to approximate emitted dose is to determine how much powderleaves its container, e.g., capture or blister, upon actuation of a drypowder inhaler (DPI). This takes into account the percentage leaving thecapsule, but does not take into account any powder depositing on theDPI. The emitted powder mass is the difference in the weight of thecapsule with the dose before inhaler actuation and the weight of thecapsule after inhaler actuation. This measurement can also be called thecapsule emitted powder mass (CEPM) or sometimes termed “shot-weight”.

A Multi-Stage Liquid Impinger (MSLI) is another device that can be usedto measure fine particle fraction. The Multi-Stage Liquid Impingeroperates on the same principles as the ACI, although instead of eightstages, MSLI has five. Additionally, each MSLI stage consists of anethanol-wetted glass frit instead of a solid plate. The wetted stage isused to prevent particle bounce and re-entrainment, which can occur whenusing the ACI.

The geometric particle size distribution can be measured for therespirable dry powder after being emitted from a dry powder inhaler(DPI) by use of a laser diffraction instrument such as the MalvernSpraytec. With the inhaler adapter in the closed-bench configuration, anairtight seal is made to the DPI, causing the outlet aerosol to passperpendicularly through the laser beam as an internal flow. In this way,known flow rates can be drawn through the DPI by vacuum pressure toempty the DPI. The resulting geometric particle size distribution of theaerosol is measured by the photodetectors with samples typically takenat 1000 Hz for the duration of the inhalation and the DV50, GSD, FPF<5.0μm measured and averaged over the duration of the inhalation.

The invention also relates to a respirable dry powder or respirable dryparticles produced using any of the methods described herein.

The respirable dry particles of the invention can also be characterizedby the chemical stability of the salts or the excipients that therespirable dry particles comprise. The chemical stability of theconstituent salts can affect important characteristics of the respirableparticles including shelf-life, proper storage conditions, acceptableenvironments for administration, biological compatibility, andeffectiveness of the salts. Chemical stability can be assessed usingtechniques well known in the art. One example of a technique that can beused to assess chemical stability is reverse phase high performanceliquid chromatography (RP-HPLC). Respirable dry particles of theinvention include salts that are generally stable over a long period oftime.

If desired, the respirable dry particles and dry powders describedherein can be further processed to increase stability. An importantcharacteristic of pharmaceutical dry powders is whether they are stableat different temperature and humidity conditions. Unstable powders willabsorb moisture from the environment and agglomerate, thus alteringparticle size distribution of the powder.

Excipients, such as maltodextrin, may be used to create more stableparticles and powders. For example, maltodextrin may act as an amorphousphase stabilizer and inhibit the components from converting from anamorphous to crystalline state. Alternatively, a post-processing step tohelp the particles through the crystallization process in a controlledway (e.g., on the baghouse at elevated humidity) can be employed withthe resultant powder potentially being further processed to restoretheir dispersibility if agglomerates formed during the crystallizationprocess, such as by passing the particles through a cyclone to breakapart the agglomerates. In an aspect of the example, the baghouse is aproduct filter. Another possible approach is to optimize around processconditions that lead to manufacturing particles that are morecrystalline and therefore more stable. Another approach is to usedifferent excipients, or different levels of current excipients toattempt to manufacture more stable forms of the salts.

Therapeutic Use and Methods

Preferably, the divalent metal cation of the dry powder formulationsdescribed herein does not produce prophylactic and/or therapeuticefficacy. Preferably, the prophylactic and/or therapeutic effect is abiological activity selected from anti-bacterial activity, anti-viralactivity, anti-inflammatory activity, mucociliary clearance, andcombinations thereof. Whether a metal cation, on its own, has such aprophylactic and/or therapeutic effect can be evaluated using one ormore in vivo models described herein or well known in the art.

For example, as used herein, a divalent metal cation does not haveanti-bacterial activity when it results in less than 50% reduction incolony forming units recovered from the lung in the mouse model ofbacterial pneumonia (in vivo pneumonia mouse model). As used herein, adivalent metal cation does not have anti-viral activity when it resultsin less than 50% reduction in nasal wash viral titer in a ferret modelof influenza infection (in vivo influenza ferret model). As used herein,a divalent metal cation does not have anti-inflammatory activity when itresults in less than 15% reduction in neutrophils recovered from thelung in the tobacco smoke mouse model of COPD (in vivo pneumonia mousemodel). As used herein, a divalent metal cation does not havemucociliary clearance (MCC) activity when a formulation administered toa sheep results in less than 12% of mucus at the one hour point incomparison to baseline measurements. The models and tests are runsubstantially as described herein or known in the art.

In Vivo Pneumonia Mouse Model

S. pneumoniae can be prepared by growing cultures on tryptic soy agar(TSA) blood plates overnight at 37° C. plus 5% CO₂. Single colonies areresuspended to an OD600˜0.3 in sterile PBS and subsequently diluted 1:4in sterile PBS (˜2×107 Colony forming units (CFU)/mL). Mice can beinfected with 504 of bacterial suspension (˜1×106 CFU) by intratrachealinstillation while under anesthesia. Mice (e.g., C57BL6) may be exposedto aerosolized liquid formulations in a whole-body exposure system e.g.,using either a high output nebulizer or Pari LC Sprint nebulizerconnected to a pie chamber cage that individually holds up to 11animals. Mice can be treated with dry powder formulations describedherein before infection with S. pneumoniae. As a control, animals may beexposed to a similar amount of placebo (e.g., 100% leucine powder).Twenty-four hours after infection mice are euthanized e.g., bypentobarbital injection and lungs are collected and homogenized insterile PBS. Lung homogenate samples are serially diluted in sterile PBSand plated on TSA blood agar plates. CFU are enumerated the followingday.

In Vivo COPD Mouse Model

Chronic obstructive pulmonary disease (COPD) is a progressive diseaseassociated with impaired pulmonary function and it primarily occurs as aresult of cigarette smoking COPD subjects are further susceptible toexacerbations that are often associated with an infectious agent andacute inflammation. These exacerbations lead to further decline in lungfunction, which in turn drives the increased frequency and severity ofsubsequent exacerbations. Animal models for COPD have been developed,e.g., a model of tobacco smoke (TS) exposure leading to acute airwayinflammation. (Churg, A. et al. Am J Physiol Lung Cell Mol Physiol294(4):L612-631, 2008; Churg, A. and J. L. Wright, Proc Am Thorac Soc6(6):550-552, 2009; Fox, J. C. and Fitzgerald M. R., Curr Opin Pharmacol9(3):231-242, 2009). The inflammation characteristic of the model ismarked by increases in macrophages and neutrophils, with modestincreases observed in lymphocytes and epithelial cells.

For example, a 4-day TS exposure model may be employed as shown inSchematic 1.

Mice (e.g., C57BL6/J) are exposed to TS for up to 45 minutes per day onfour successive days by whole body exposure. On each day of TS exposure,mice may be treated with dry powder formulations described herein 1 hourbefore and 6 hours after TS exposure or with placebo control (e.g., 100%leucine). Dosing may be performed using a whole body exposure system anda capsule based delivery system. A p38 MAP kinase inhibitor ADS110836may be used as a reference agent (positive control) (see, PCT Publ. No.WO2009/098612) that can be administered by an intranasal route. Animalsmay be euthanized by intra-peritoneal barbiturate anaesthetic overdose24 hours after the final exposure to either air (sham) or TS on day 5. Abronchoalveolar lavage (BAL) can be performed using 0.4 mL of phosphatebuffered saline (PBS). Cells recovered from the BAL are enumerated anddifferential cell counts carried out using cytospin prepared slides.Inflammatory cell counts in the BAL fluid of animals exposed to TS for 4days are determined. Dosing may be calculated by collecting samples fromthe pie cage system onto a glass fiber filter at 1 LPM. The aerosolcollected onto the filter is recovered and the cation concentration isdetermined by HPLC to determine the aerosol concentration of cation(Ec). The estimated dose level (DL) is given by the equation:DL=Ec·RMV·T/BW, where RMV is the respiratory minute volume of the animal(0.21 LPM), T is the exposure time, and BW is the body weight of theanimal in kg. The resulting estimated dose is then adjusted for therespirable fraction of the aerosol, which is determined based on thefine particle fraction (FPF; % mass less than 5.6 μm).

In Vivo Influenza Ferret Model

Ferrets infected with influenza typically show increases in bodytemperature within 2 days of infection, drop body weight over the courseof the study and show clinical signs of infection such as lethargy andsneezing. These changes coincide with an increase in influenza viraltiters shed from the nasal cavity and increases in nasal inflammation.Dry powder formulations described herein and placebo control powders areaerosolized, e.g., using a Palas Rotating Brush Generator 1000 solidparticle disperser (RBG, Palas GmbH, Karlsruhe, Germany). Ferrets areexposed to the dry powder formulations described herein and the severityof infection is evaluated over time. Each formulation may be dispersedin a nose-only exposure system 1 hour before infection, 4 hours afterinfection and then BID for 4 days (d1-4). The study may be terminated onday 10. Body temperatures are determined, e.g., twice a day, beginningon day 0 of the study. On study day −4, ferrets can be implanted with amicrochip subcutaneously, e.g., in the right rear flank and another inthe shoulder for redundancy. The transponder chip (e.g., IPTT-300Implantable Programmable Temperature and Identification Transponder; BioMedic Data Systems, Inc, Seaford, Delaware 19973) allows for ferretidentification and provides subcutaneous body temperature datathroughout the study using e.g., a BMDS electronic proximity reader wand(WRS-6007; Biomedic Data Systems Inc, Seaford, Delaware). Subcutaneousbody temperatures taken on day −3 to −1 are used as baselinetemperatures and used to calculate the change from baseline for eachanimal over the course of the study.

In Vivo Mucociliary Clearance (MCC) Sheep Model

Mucociliary clearance (MCC) can be evaluated in healthy sheep bymeasurement of the clearance of pulmonary Tc99m-labeled sulfur colloidaerosols that is delivered by inhalation. Immediately following theexposure to the test-formulation (the formulation that is tested foractivity), the radio-labeled sulfur colloid aerosol is delivered to thesheep via the same aerosol delivery system and MCC is determined via thecollection of serial images. The sheep mucociliary clearance model is awell established model with vehicle clearance typically measuringapproximately 5-10% at 60 minutes after delivery of the radioactiveaerosol (see, for example, Coote et al., 2009, JEPT 329:769-774). It isknown in the art that average clearance measurements greater than about10% at 60 minutes post baseline indicate enhanced clearance in themodel.

For example, a Pari LC jet nebulizer exposure system may be used todeliver the test-formulation. The nebulizer is connected to a dosimetersystem consisting of a solenoid valve and a source of compressed air (20psi). The output of the nebulizer is connected to a T-piece, with oneend attached to a respirator (Harvard Apparatus Inc., Holliston, Mass.).The system is activated for 1 second at the onset of the inspiratorycycle of the respirator, which is set at an inspiratory/expiratory ratioof 1:1 and a rate of 20 breaths/minute. A tidal volume of 300 ml is usedto deliver the nebulized formulation. The nebulizer was filled with thetest-formulation and run to dryness. Alternatively, the test-formulationcan be delivered with a rotating brush generator (RBG1000, Palas)instead of the nebulizer to generate the dry powder aerosol. Aerosolizedtechnetium labeled sulfur colloid (99 mTC-SC) is delivered immediatelyafter the test-formulation. Animals are conscious, supported in a mobilerestraint, intubated with a cuffed endotracheal tube and maintainedconscious for the duration of the study.

After 99m TC-SC nebulization, the animals are immediately extubated andpositioned in their natural upright position underneath a gamma camera(Dyna Cam, Picker Corp., Nothford, Conn.) so that the field of image isperpendicular to the animals' spinal cord. After acquisition of abaseline image, serial images are obtained at 5 min intervals for thefirst hour. All images are obtained and stored in the computer foranalysis. An area of interest is traced over the image corresponding tothe right lung of the animals, and counts are recorded. The left lung isexcluded from analysis because its corresponding image is superimposedover the stomach and counts could be affected by swallowed radiolabeledmucus. The counts are corrected for decay and clearance expressed as thepercentage reduction of radioactivity present from the baseline image.

The dose delivered for test-formulations is measured in-vitro with abreathing simulator system drawing the inspiratory flow through filtersamples collected at the distal end of a tracheal tube. For example, 10filter samples of 1.5 minutes each are assayed for deposited divalentmetal cation by HPLC and the average rate of divalent metal cationdeposition was determined. From this the dose delivered in 15 minutes toa 50 kg sheep is calculated. These measured doses correspond to the dosedelivered from the distal end of the tracheal tube to the sheep duringtreatment.

The respirable dry powders and respirable dry particles of the presentinvention are for administration to the respiratory tract.Administration to the respiratory tract can be for local activity of thedelivered pharmaceutically active agent or for systemic activity. Forexample, the respirable dry powders can be administered to the nasalcavity or upper airway to provide, for example, anti-inflammatory,anti-viral, or anti-bacterial activity to the nasal cavity or upperairway. The respirable dry powders can be administered to the deep lungto provide local activity in the lung or for absorption into thesystemic circulation. Systemic delivery of certain pharmaceuticallyactive agents via the lung is particularly advantageous for agents thatundergo substantial first pass metabolism (e.g., in the liver) followingoral administration.

The respirable dry powders and respirable dry particles of the presentinvention may also be administered to the buccal cavity. Administrationto the buccal cavity can be for local activity of the deliveredpharmaceutically active agent or for systemic activity. For exaple, therespirable dry powders can be administered to the buccal cavity toprovide, for example, anti-inflammatory, anti-viral, or anti-bacterialactivity to the buccal cavity.

The dry powders and dry particles of the invention can be administeredto a subject in need thereof for systemic delivery of a pharmaceuticallyactive agent, such as to treat an infectious disease or metabolicdisease.

The dry powders and dry particles of the invention can be administeredto a subject in need thereof for the treatment of respiratory (e.g.,pulmonary) diseases, such as respiratory syncytial virus infection,idiopathic fibrosis, alpha-1 antitrypsin deficiency, asthma, airwayhyperresponsiveness, seasonal allergic allergy, brochiectasis, chronicbronchitis, emphysema, chronic obstructive pulmonary disease, cysticfibrosis and the like, and for the treatment and/or prevention of acuteexacerbations of these chronic diseases, such as exacerbations caused byviral infections (e.g., influenza virus, parainfluenza virus,respiratory syncytial virus, rhinovirus, adenovirus, metapneumovirus,coxsackie virus, echo virus, corona virus, herpes virus,cytomegalovirus, and the like), bacterial infections (e.g.,Streptococcus pneumoniae, which is commonly referred to as pneumococcus,Staphylococcus aureus, Burkholderis ssp., Streptococcus agalactiae,Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiellapneumoniae, Escherichia coli, Pseudomonas aeruginosa, Moraxellacatarrhalis, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionellapneumophila, Serratia marcescens, Mycobacterium tuberculosis, Bordetellapertussis, and the like), fungal infections (e.g., Histoplasmacapsulatum, Cryptococcus neoformans, Pneumocystis jiroveci, Coccidioidesimmitis, and the like) or parasitic infections (e.g., Toxoplasma gondii,Strongyloides stercoralis, and the like), or environmental allergens andirritants (e.g., aeroallergens, including pollen and cat dander,airborne particulates, and the like).

The dry powders and dry particles of the invention can be administeredto a subject in need thereof for the treatment and/or prevention and/orreducing contagion of infectious diseases of the respiratory tract, suchas pneumonia (including community-acquired pneumonia, nosocomialpneumonia (hospital-acquired pneumonia, HAP; health-care associatedpneumonia, HCAP), ventilator-associated pneumonia (VAP)),ventilator-associated tracheobronchitis (VAT), bronchitis, croup (e.g.,postintubation croup, and infectious croup), tuberculosis, influenza,common cold, and viral infections (e.g., influenza virus, parainfluenzavirus, respiratory syncytial virus, rhinovirus, adenovirus,metapneumovirus, coxsackie virus, echo virus, corona virus, herpesvirus, cytomegalovirus, and the like), bacterial infections (e.g.,Streptococcus pneumoniae, which is commonly referred to as pneumococcus,Staphylococcus aureus, Streptococcus agalactiae, Haemophilus influenzae,Haemophilus parainfluenzae, Klebsiella pneumoniae, Escherichia coli,Pseudomonas aeruginosa, Moraxella catarrhalis, Chlamydophila pneumoniae,Mycoplasma pneumoniae, Legionella pneumophila, Serratia marcescens,Mycobacterium tuberculosis, Bordetella pertussis, and the like), fungalinfections (e.g., Histoplasma capsulatum, Cryptococcus neoformans,Pneumocystis jiroveci, Coccidioides immitis, and the like) or parasiticinfections (e.g., Toxoplasma gondii, Strongyloides stercoralis, and thelike), or environmental allergens and irritants (e.g., aeroallergens,airborne particulates, and the like).

In some aspects, the invention provides a method for treating apulmonary disease, such as asthma, airway hyperresponsiveness, seasonalallergic allergy, bronchiectasis, chronic bronchitis, emphysema, chronicobstructive pulmonary disease, cystic fibrosis and the like, comprisingadministering to the respiratory tract of a subject in need thereof aneffective amount of respirable dry particles or dry powder, as describedherein.

In other aspects, the invention provides a method for the treatment orprevention of acute exacerbations of a chronic pulmonary disease, suchas asthma, airway hyperresponsiveness, seasonal allergic allergy,bronchiectasis, chronic bronchitis, emphysema, chronic obstructivepulmonary disease, cystic fibrosis and the like, comprisingadministering to the respiratory tract of a subject in need thereof aneffective amount of respirable dry particles or dry powder, as describedherein.

In other aspects, the invention provides a method for treating,preventing and/or reducing contagion of an infectious disease of therespiratory tract, comprising administering to the respiratory tract ofa subject in need thereof an effective amount of respirable dryparticles or dry powder, as described herein.

In some aspects, the invention provides a method for the treatment orprevention of cardiovascular disease, auto-immune disorders, transplantrejections, autoimmune disorders, allergy-related asthma, infections,and cancer. For example, the invention provides a method for thetreatment or prevention of postmenopausal osteoporosis,cryopyrin-associated periodic syndromes (CAPS), paroxysmal nocturnalhemoglobinuria, psoriasis, rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, multiple sclerosis, and macular degeneration.For example, dry powders or dry particles of the invention areco-formulated or blended with therapeutic antibodies as describedherein. The co-formulated or blended dry powders may then beadministered to a subject in need of therapy or prevention.

In certain aspects, the invention provides a method for the treatment orprevention of cancer such as acute myeloid leukemia, B cell leukemia,non-Hodgkin's lymphoma, breast cancer (e.g., with HER2/neuoverexpression), glioma, squamous cell carcinomas, colorectal carcinoma,anaplastic large cell lymphoma (ALCL), Hodgkin lymphoma, head and neckcancer, acute myelogenous leukemia (AML), melanoma, and chroniclymphocytic leukemia (CLL). Alternatively or in addition, the inventionprovides a method for the treatment or prevention of cancer byanti-angiogenic cancer therapy. For example, dry powders or dryparticles of the invention are co-formulated or blended with therapeuticantibodies as described herein. Therapeutic antibodies can becancer-specific antibodies, such as a humanized monoclonal antibody,e.g., gemtuzumab, alemtuzumab, trastuzumab, nimotuzumab, bevacizumab, ora chimeric monoclonal antibody, e.g., rituximab and cetuximab. Theco-formulated or blended dry powders may then be administered to asubject in need of therapy or prevention.

In certain aspects, the invention provides a method for the treatment orprevention of inflammation such as rheumatoid arthritis, Crohn'sdisease, ulcerative Colitis, acute rejection of kidney transplants,moderate-to-severe allergic asthma. For example, dry powders or dryparticles of the invention are co-formulated or blended with therapeuticantibodies as described herein. Therapeutic antibodies can beinflammation-specific antibodies, such as chimeric monoclonalantibodies, e.g., infliximab, basiliximab, humanized monoclonalantibodies, e.g., daclizumab, omalizumab, or human antibodies, e.g.,adalimumab. The co-formulated or blended dry powders may then beadministered to a subject in need of therapy or prevention.

In certain aspects, the invention provides a method for the treatment orprevention of RSV infections in children. For example, dry powders ordry particles of the invention are co-formulated or blended withtherapeutic antibodies as described herein. Therapeutic antibodies canbe RSV infection-specific antibodies, such as the humanized monoclonalantibody palivizumab which inhibits an RSV fusion (F) protein. Theco-formulated or blended dry powders may then be administered to asubject in need of RSV infection therapy or prevention.

In certain aspects, the invention provides a method for the treatment orprevention of diabetes. For example, dry powders or dry particles of theinvention are co-formulated or blended with insulin as described herein.The co-formulated or blended dry powders may then be administered to asubject in need of insulin therapy or prevention.

The respirable dry particles and dry powders can be administered to therespiratory tract of a subject in need thereof using any suitablemethod, such as instillation techniques, and/or an inhalation device,such as a dry powder inhaler (DPI) or metered dose inhaler (MDI). Anumber of DPIs are available, such as, the inhalers disclosed is U.S.Pat. Nos. 4,995,385 and 4,069,819, Spinhaler® (Fisons, Loughborough,U.K.), Rotahalers®, Diskhaler® and Diskus® (GlaxoSmithKline, ResearchTriangle Technology Park, North Carolina), FlowCapss® (Hovione, Loures,Portugal), Inhalators® (Boehringer-Ingelheim, Germany), Aerolizer®(Novartis, Switzerland), RS-01 (Plastiape, Italy) and others known tothose skilled in the art.

Generally, inhalation devices (e.g., DPIs) are able to deliver a maximumamount of dry powder or dry particles in a single inhalation, which isrelated to the capacity of the blisters, capsules (e.g., size 000, 00,0E, 0, 1, 2, 3, and 4, with respective volumetric capacities of 1.37 ml,950 μl, 770 μl, 680 μl, 480 μl, 360 μl, 270 μl, and 200 μl) or othermeans that contain the dry particles or dry powders within the inhaler.Accordingly, delivery of a desired dose or effective amount may requiretwo or more inhalations. Preferably, each dose that is administered to asubject in need thereof contains an effective amount of respirable dryparticles or dry powder and is administered using no more than about 4inhalations. For example, each dose of respirable dry particles or drypowder can be administered in a single inhalation or 2, 3, or 4inhalations. The respirable dry particles and dry powders are preferablyadministered in a single, breath-activated step using a breath-activatedDPI. When this type of device is used, the energy of the subject'sinhalation both disperses the respirable dry particles and draws theminto the respiratory tract.

The respirable dry particles or dry powders can be delivered byinhalation to a desired area within the respiratory tract, as desired.It is well-known that particles with an aerodynamic diameter of about 1micron to about 3 microns, can be delivered to the deep lung. Largeraerodynamic diameters, for example, from about 3 microns to about 5microns can be delivered to the central and upper airways.

For dry powder inhalers, oral cavity deposition is dominated by inertialimpaction and so characterized by the aerosol's Stokes number (DeHaan etal. Journal of Aerosol Science, 35 (3), 309-331, 2003). For equivalentinhaler geometry, breathing pattern and oral cavity geometry, the Stokesnumber, and so the oral cavity deposition, is primarily affected by theaerodynamic size of the inhaled powder. Hence, factors which contributeto oral deposition of a powder include the size distribution of theindividual particles and the dispersibility of the powder. If the MMADof the individual particles is too large, e.g., above 5 μm, then anincreasing percentage of powder will deposit in the oral cavity.Likewise, if a powder has poor dispersibility, it is an indication thatthe particles will leave the dry powder inhaler and enter the oralcavity as agglomerates. Agglomerated powder will perform aerodynamicallylike an individual particle as large as the agglomerate, therefore evenif the individual particles are small (e.g., MMAD of 5 microns or less),the size distribution of the inhaled powder may have an MMAD of greaterthan 5 μm, leading to enhanced oral cavity deposition.

Therefore, it is desirable to have a powder in which the particles aresmall (e.g., MMAD of 5 microns or less, e.g., between 1 to 5 microns),and are highly dispersible (e.g., 1/4 bar or alternatively, 0.5/4 bar of2.0, and preferably less than 1.5). More preferably, the respirable drypowder is comprised of respirable dry particles with an MMAD between 1to 4 microns or 1 to 3 microns, and have a 1/4 bar less than 1.4, orless than 1.3, and more preferably less than 1.2.

The absolute geometric diameter of the particles measured at 1 bar usingthe HELOS system is not critical provided that the particle's envelopedensity is sufficient such that the MMAD is in one of the ranges listedabove, wherein MMAD is VMGD times the square root of the envelopedensity (MMAD=VMGD*sqrt(envelope density)). If it is desired to delivera high unit dose of salt using a fixed volume dosing container, then,particles of higher envelop density are desired. High envelope densityallows for more mass of powder to be contained within the fixed volumedosing container. Preferable envelope densities are greater than 0.1g/cc, greater than 0.25 g/cc, greater than 0.4 g/cc, greater than 0.5g/cc, and greater than 0.6 g/cc.

The respirable dry powders and particles of the invention can beemployed in compositions suitable for drug delivery via the respiratorysystem. For example, such compositions can include blends of therespirable dry particles of the invention and one or more other dryparticles or powders, such as dry particles or powders that contain anactive agent, or that consist of or consist essentially of one or morepharmaceutically acceptable excipients.

Respirable dry powders and dry particles suitable for use in the methodsof the invention can travel through the upper airways (i.e., theoropharynx and larynx), the lower airways, which include the tracheafollowed by bifurcations into the bronchi and bronchioli, and throughthe terminal bronchioli which in turn divide into respiratory bronchiolileading then to the ultimate respiratory zone, the alveoli or the deeplung. In one embodiment of the invention, most of the mass of respirabledry powders or particles deposit in the deep lung. In another embodimentof the invention, delivery is primarily to the central airways. Inanother embodiment, delivery is to the upper airways.

The respirable dry particles or dry powders of the invention can bedelivered by inhalation at various parts of the breathing cycle (e.g.,laminar flow at mid-breath). An advantage of the high dispersibility ofthe dry powders and dry particles of the invention is the ability totarget deposition in the respiratory tract. For example, breathcontrolled delivery of nebulized solutions is a recent development inliquid aerosol delivery (Dalby et al. in Inhalation Aerosols, edited byHickey 2007, p. 437). In this case, nebulized droplets are released onlyduring certain portions of the breathing cycle. For deep lung delivery,droplets are released in the beginning of the inhalation cycle, whilefor central airway deposition, they they are released later in theinhalation.

The highly dispersible powders of the invention can provide advantagesfor targeting the timing of drug delivery in the breathing cycle andalso location in the human lung. Because the respirable dry powders ofthe invention can be dispersed rapidly, such as within a fraction of atypical inhalation maneuver, the timing of the powder dispersal can becontrolled to deliver an aerosol at specific times within theinhalation.

With a highly dispersible powder, the complete dose of aerosol can bedispersed at the beginning portion of the inhalation. While thepatient's inhalation flow rate ramps up to the peak inspiratory flowrate, a highly dispersible powder will begin to disperse already at thebeginning of the ramp up and could completely disperse a dose in thefirst portion of the inhalation. Since the air that is inhaled at thebeginning of the inhalation will ventilate deepest into the lungs,dispersing the most aerosol into the first part of the inhalation ispreferable for deep lung deposition. Similarly, for central deposition,dispersing the aerosol at a high concentration into the air which willventilate the central airways can be achieved by rapid dispersion of thedose near the mid to end of the inhalation. This can be accomplished bya number of mechanical and other means such as a switch operated bytime, pressure or flow rate which diverts the patient's inhaled air tothe powder to be dispersed only after the switch conditions are met.

Aerosol dosage, formulations and delivery systems may be selected for aparticular therapeutic application, as described, for example, in Gonda,I. “Aerosols for delivery of therapeutic and diagnostic agents to therespiratory tract,” in Critical Reviews in Therapeutic Drug CarrierSystems, 6: 273-313 (1990); and in Moren, “Aerosol Dosage Forms andFormulations,” in Aerosols in Medicine, Principles, Diagnosis andTherapy, Moren, et al., Eds., Esevier, Amsterdam (1985).

Suitable dosing to provide the desired therapeutic effect can bedetermined by a clinician based on the severity of the condition (e.g.,infection), overall well being of the subject and the subject'stolerance to respirable dry particles and dry powders and otherconsiderations. Based on these and other considerations, a clinician candetermine appropriate doses and intervals between doses. Generally,respirable dry particles and dry powders are administered once, twice orthree times a day, as needed.

If desired or indicated, the respirable dry particles and dry powdersdescribed herein can be administered with one or more other therapeuticagents. The other therapeutic agents can be administered by any suitableroute, such as orally, parenterally (e.g., intravenous, intraarterial,intramuscular, or subcutaneous injection), topically, by inhalation(e.g., intrabronchial, intranasal or oral inhalation, intranasal drops),rectally, vaginally, and the like. The respirable dry particles and drypowders can be administered before, substantially concurrently with, orsubsequent to administration of the other therapeutic agent. Preferably,the respirable dry particles and dry powders and the other therapeuticagent are administered so as to provide substantial overlap of theirpharmacologic activities.

Another advantage provided by the respirable dry powders and respirabledry particles described herein, is that dosing efficiency can beincreased as a result of hygroscopic growth of particles inside thelungs, due to particle moisture growth. The propensity of the partiallyamorphous compositions of the invention to take up water at elevatedhumidities can also be advantageous with respect to their depositionprofiles in vivo. Due to their rapid water uptake at high humidities,these powder formulations can undergo hygroscopic growth due to theabsorbance of water from the humid air in the respiratory tract as theytransit into the lungs. This can result in an increase in theireffective aerodynamic diameters during transit into the lungs, whichwill further facilitate their deposition in the airways.

EXEMPLIFICATION Methods

Geometric or Volume Diameter.

Volume median diameter (×50), which may also be referred to as volumemedian geometric diameter (VMGD), was determined using a laserdiffraction technique. The equipment consisted of a HELOS diffractometerand a RODOS dry powder disperser (Sympatec, Inc., Princeton, N.J.). TheRODOS disperser applies a shear force to a sample of particles,controlled by the regulator pressure (typically set at 1.0 bar withmaximum orifice ring pressure) of the incoming compressed dry air. Thepressure settings may be varied to vary the amount of energy used todisperse the powder. For example, the regulator pressure may be variedfrom 0.2 bar to 4.0 bar. Powder sample is dispensed from a microspatulainto the RODOS funnel. The dispersed particles travel through a laserbeam where the resulting diffracted light pattern produced is collected,typically using an R1 lens, by a series of detectors. The ensemblediffraction pattern is then translated into a volume-based particle sizedistribution using the Fraunhofer diffraction model, on the basis thatsmaller particles diffract light at larger angles. Using this methodgeometric standard deviation (GSD) for the volume mean geometricdiameter was also determined.

Fine Particle Fraction.

The aerodynamic properties of the powders dispersed from an inhalerdevice were assessed with a Mk-II 1 ACFM Andersen Cascade Impactor(Copley Scientific Limited, Nottingham, UK). The instrument was run incontrolled environmental conditions of 18 to 25° C. and relativehumidity (RH) between 25 and 35%. The instrument consists of eightstages that separate aerosol particles based on inertial impaction. Ateach stage, the aerosol stream passes through a set of nozzles andimpinges on a corresponding impaction plate. Particles having smallenough inertia will continue with the aerosol stream to the next stage,while the remaining particles will impact upon the plate. At eachsuccessive stage, the aerosol passes through nozzles at a highervelocity and aerodynamically smaller particles are collected on theplate. After the aerosol passes through the final stage, a filtercollects the smallest particles that remain. Gravimetric or analyticanalysis can then be performed to determine the particle sizedistribution.

The impaction technique utilized allowed for the collection of eightseparate powder fractions. The capsules (Capsugel, Greenwood, S.C.) werehalf-filled with approximately 20 or 50 mg powder and placed in ahand-held, breath-activated dry powder inhaler (DPI) device, the highresistance RS-01 DPI (Plastiape, Osnago, Italy). In some instances, thecapsules were filled as much as necessary to fit the desired mass ofpowder into the capsule. The capsule was punctured and the powder wasdrawn through the cascade impactor operated at a flow rate of 60.0 L/minfor 2.0 s. At this flow rate, the calibrated cut-off diameters for theeight stages are 8.6, 6.5, 4.4, 3.3, 2.0, 1.1, 0.5 and 0.3 microns. Thefractions were collected by placing filters in the apparatus anddetermining the amount of powder that impinged on them by gravimetricmeasurements. The fine particle fraction of the total dose of powder(FPF_TD) less than or equal to an effective cut-off aerodynamic diameterwas calculated by dividing the powder mass recovered from the desiredstages of the impactor by the total particle mass in the capsule.Results are reported as the fine particle fraction of less than 4.4microns (FPF<4.4 microns), as well as mass median aerodynamic diameter(MMAD) and GSD calculated from the FPF trend across stages. The fineparticle fraction can alternatively be calculated relative to therecovered or emitted dose of powder by dividing the powder massrecovered from the desired stages of the impactor by the total powdermass recovered.

Tap Density.

Tap density was measured using a modified method requiring smallerpowder quantities, following USP <616> with the substitution of a 1.5 ccmicrocentrifuge tube (Eppendorf AG, Hamburg, Germany) or a 0.3 ccsection of a disposable serological polystyrene micropipette (GrenierBio-One, Monroe, N.C.) with polyethylene caps (Kimble Chase, Vineland,N.J.) to cap both ends and hold the powder. Instruments for measuringtap density, known to those skilled in the art, include but are notlimited to the Dual Platform Microprocessor Controlled Tap DensityTester (Vankel, Cary, N.C.) or a SOTAX Tap Density Tester model TD2(Horsham, Pa.). Tap density is a standard measure of the envelope massdensity. The envelope mass density of an isotropic particle is definedas the mass of the particle divided by the minimum spherical envelopevolume within which it can be enclosed.

Bulk Density.

Bulk density was estimated prior to tap density measurement by dividingthe weight of the powder by the volume of the powder, as estimated usingthe volumetric measuring device.

Example 1 Production and Characterication of Divalent Cationic Powders

Several powders of the invention were produced by spray dryinghomogenous particles. The composition of these powders is shown in Table1.

TABLE 1 Composition of divalent cation dry powders. % % Additional %Salt Excipient component Additional load load (e.g., drug, componentForm Salt (w/w) Excipient (w/w) 2^(nd) salt) load (w/w) I Magnesium 5N/A 0 albuterol 95 sulfate II Calcium 7 Maltodextrin 43 ciprofloxacin 50sulfate III Magnesium 15 Maltodextrin 35 tobramycin 50 sulfate IVCalcium 40 Maltodextrin 10 ciprofloxacin 50 sulfate V Magnesium 58.3Leucine 37.5 Sodium 4.2 lactate chloride VI Magnesium 9 Maltodextrin90.9 Tiotropium .113 lactate bromide (TioB) VII Magnesium 10 Mannitol 90N/A N/A lactate VIII Magnesium 10 Maltodextrin 90 N/A N/A lactate IXMagnesium 10 Leucine 90 N/A N/A sulfate X Magnesium 10 Leucine 90 N/AN/A lactate

Materials used in the following Examples and their sources are listedbelow. Calcium sulfate dihydrate, magnesium lactate, magnesium sulfate,sodium chloride, L-leucine, maltodextrin, albuterol sulfate,ciprofloxacin hydrochloride and tobramycin were obtained fromSigma-Aldrich Co. (St. Louis, Mo.) or Spectrum Chemicals (Gardena,Calif.). Ultrapure water was from a water purification system (MilliporeCorp., Billerica, Mass.).

Spray drying homogenous particles requires that the ingredients ofinterest be solubilized in solution or suspended in a uniform and stablesuspension. Most of the materials mentioned above are sufficientlywater-soluble to prepare suitable spray drying solutions (see Table 2).However, calcium sulfate dihydrate has a low solubility in water. As aresult of this low solubility, formulation feedstock development workwas necessary to prepare solutions or suspensions that could be spraydried. These solutions or suspensions included combinations of salts andantibiotic, steroid or beta agonist in an appropriate solvent, typicallywater.

TABLE 2 Mono- and divalent cation salt solubilities Water solubility atSalt 20-30° C., 1 bar Magnesium carbonate 4.5 in 100 parts² Magnesiumcarbonate hydroxide Soluble in 3300 parts of CO₂ free water¹ Magnesiumchloride Hexahydrate, 1 g/0.6 mL¹ Magnesium citrate Partially soluble incold water³ Magnesium sulfate Heptahydrate, 71 g/100 mL¹ Potassiumchloride 1 g/2.8 mL¹ Potassium citrate Monohydrate, 1 g/0.65 mL¹ Sodiumascorbate 62 g/100 mL¹ Sodium bicarbonate Soluble in 10 parts¹ Sodiumcarbonate Soluble in 3.5 parts¹ Sodium chloride 1 g/2.8 mL¹ Sodiumcitrate Dihydrate, soluble in 1.3 parts¹ Sodium lactate Commerciallyavailable as 70-80% in water¹ Dibasic sodium phosphate Soluble in ~8parts¹ Sodium propionate 1 g/~1 mL¹ Sodium sulfate Soluble in 3.6 parts¹¹O'Neil, Maryadele J. The Merck Index: an Encyclopedia of Chemicals,Drugs, and Biologicals. 14th ed. Whitehouse Station, N.J.: Merck, 2006.

For the spray drying process, the salts, excipients and other drugs weredissolved or suspended in a solvent (e.g., water). The solidsconcentration (w/v) was chosen dependent on the solubility of thedifferent components. For the calcium sulfate formulation, aconcentration of 4 mg/mL was appropriate, given the limited solubilityof calcium sulfate: 2 mg/mL. In addition, when preparing spray dryingsolutions, the water weight of the hydrated starting material must beaccounted for. The ratios used for formulations were based on themolecular weight of the anhydrous salts. For calcium sulfate, thedihydrate form is more readily available than the anhydrous form. Thisrequired an adjustment in the ratios originally calculated, using amultiplier to correlate the molecular weight of the anhydrous salt withthe molecular weight of the hydrate. For example, the molecular weightof anhydrous calcium sulfate is 136.14 g/mol and the dihydrate molecularweight is 172.172 g/mol, so a multiplier of 1.26 will be used tocalculate the amount of calcium sulfate dihydrate weighed. For a 5 gtotal solids concentration where 40% is calcium sulfate, the anhydrousweighed amount of calcium sulfate would be 2 g; instead using themultiplier, a weighed amount of 2.529 g was targeted.

Dry powders were prepared by spray drying on a Büchi B-290 Mini SprayDryer (BUCHI Labortechnik AG, Flawil, Switzerland) with powdercollection from either a standard or High Performance cyclone. Thesystem used the Büchi B-296 dehumidifier to ensure stable temperatureand humidity of the air used to spray dry. Furthermore, when therelative humidity in the room exceeded 30% RH, an external LGdehumidifier (model 49007903, LG Electronics, Englewood Cliffs, N.J.)was run constantly. Atomization of the liquid feed utilized a Büchitwo-fluid nozzle with a 1.5 mm diameter. Inlet temperature of theprocess gas can range from 100° C. to 220° C. and outlet temperaturefrom 80° C. to 120° C. with a liquid feedstock flowrate of 3 mL/min to10 mL/min. Outlet temperatures can range from 50° C. to 80° C. Thetwo-fluid atomizing gas ranges from 25 mm to 45 mm (355 LPH to 831 LPH)and the aspirator rate ranges from 70% to 100% (28 m³/hr to 38 m³/hr).

The feedstock was prepared as a batch by dissolving the specific salt inultrapure water, then the excipient, and finally the drug component. Thesolution was kept agitated throughout the process until the materialswere completely dissolved in the water at room temperature.

Formulation I dry powders were produced by spray drying on the BüchiB-290 Mini Spray Dryer (BUCHI Labortechnik AG, Flawil, Switzerland) withpowder collection on a 60 mL glass vessel from a High Performancecyclone. The system used the Büchi B-296 dehumidifier and an external LGdehumidifier (model 49007903, LG Electronics, Englewood Cliffs, N.J.)was run constantly. Atomization of the liquid feed utilized a Büchitwo-fluid nozzle with a 1.5 mm diameter. The two-fluid atomizing gas wasset at 40 mm. Aspirator rate varied between 80% and 90% (32 and 35 m³h).Room air was used as the drying gas. Inlet temperature of the processgas was 180° C. and outlet temperature at 85° C. with a liquid feedstockflow rate of 6 mL/min to 7 mL/min. The solids concentration was 4.2 g/Lin ultrapure water.

Formulation II was produced using the same equipment and settings. Inlettemperature of the process gas was 180° C. and outlet temperature from85° C. to 86° C. with a liquid feedstock flow rate of 6 mL/min to 7mL/min. Aspirator rate was 90% (35 m³h). The solids concentration was 5g/L in ultrapure water.

Formulation III was produced using the same equipment and settings.Inlet temperature of the process gas was 180° C. and outlet temperaturefrom 83° C. to 84° C. with a liquid feedstock flow rate of 6 mL/min to 7mL/min. Aspirator rate was 90% (35 m³h). The solids concentration was 5g/L in ultrapure water.

Formulation IV was produced using the same equipment and settings. Inlettemperature of the process gas was 180° C. and outlet temperature from83° C. to 84° C. with a liquid feedstock flow rate of 5 mL/min to 6mL/min. Aspirator rate was 90% (35 m³h). The solids concentration was 4g/L in ultrapure water.

Formulation V was produced using the same equipment and settings. Inlettemperature of the process gas was 180° C. and outlet temperature from76° C. to 77° C. with a liquid feedstock flow rate of 6 mL/min.Aspirator rate was 90% (35 m³h). The solids concentration was 10 g/L inultrapure water.

The powders produced were characterized with regard to density anddispersibility ratio. Bulk and tapped densities were determined using aSOTAX Tap Density Tester model TD1 (Horsham, Pa.). For any given run,the entire sample was introduced to a tared 0.3 cc section of adisposable serological polystyrene micropipette (Grenier Bio-One,Monroe, N.C.) using a funnel made with weighing paper (VWRInternational, West Chester, Pa.) and the pipette section was pluggedwith polyethylene caps (Kimble Chase, Vineland, N.J.) to hold thepowder. The powder mass and initial volume (V₀) were recorded and thepipette was attached to the anvil and run according to the USP I method.For the first pass, the pippette was tapped using Tap Count 1 (500 taps)and the resulting volume V_(a) was recorded. For the second pass, TapCount 2 was used (750 taps) resulting in the new volume V_(b1). IfV_(b1)>98% of V_(a), the test was complete, otherwise Tap Count 3 wasused (1250 taps) iteratively until V_(bn)>98% of V_(bn-1). Bulk densitywas estimated prior to tap density measurement by dividing the weight ofthe powder by the volume of the powder, as estimated using thevolumetric measuring device. Calculations were made to determine thepowder bulk density (d_(B)), tap density (d_(T)), and Hausner Ratio (H),which is the tap density divided by the bulk density.

Volume median diameter was determined using a HELOS laser diffractometerand a RODOS dry powder disperser (Sympatec, Inc., Princeton, N.J.). Amicrospatula of material (approximately 5 milligrams) was introducedinto the RODOS funnel, where a shear force is applied to a sample ofparticles as controlled by the regulator pressure of the incomingcompressed dry air. The pressure settings were varied to use differentamounts of energy to disperse the powder. The regulator pressure was setat 0.2 bar, 0.5 bar, 1.0 bar, 2.0 bar and 4.0 bar, with maximum orificering pressure at each pressure. The dispersed particles traveled througha laser beam where the resulting diffracted light pattern produced iscollected, using an R1 or R3 lens, by a series of detectors. Theensemble diffraction pattern is then translated into a volume-basedparticle size distribution using the Fraunhofer diffraction model, onthe basis that smaller particles diffract light at larger angles. 1/4bar, 0.5/4 bar, 0.2/4 bar ratios were obtained by dividing averagevolume median diameter values obtained at each of 0.2 bar, 0.5 bar and1.0 bar by the volume median diameter value obtained at 4.0 bar.

Results for the density tests for Formulations I, II, III, IV and V areshown in Table 3. The tap densities for all formulations are relativelyhigh, especially for Formulation V. The bulk densities are such that theHausner ratios are rather high for all formulations, particularlyFormulation II and Formulation V. All five of the powders tested possessHausner Ratios that are described in the art as being characteristic ofpowders with extremely poor flow properties (See, e.g., USP <1174>). USP<1174> notes that dry powders with a Hausner ratio greater than 1.35 arepoor flowing powders. Flow properties and dispersibility are bothnegatively affected by particle agglomeration or aggregation. It istherefore unexpected that powders with Hasuner Ratios of 1.4 to 2.8would be highly dispersible. This strengthens the counter-intuitivenessof these formulations being highly dispersible and possessing goodaerosolization properties. In particular, Formulation V has a high tapdensity (0.78 g/cc) and a high Hausner ratio (2.79).

TABLE 3 Divalent cationic dry powder characteristics. DensityHELOS/RODOS Formu- Bulk Density Tap Density Hausner 1/4 0.5/4 0.2/4lation (g/cc) (g/cc) Ratio bar bar bar I 0.34 ± 0.06 0.47 ± 0.03 1.381.00 1.09 1.10 MgSul: Albu II 0.22 ± 0.02 0.46 ± 0.01 2.09 1.04 1.111.28 CaSul: Malto: Cipro III 0.34 ± 0.09 0.52 ± 0.02 1.53 1.05 1.10 1.20MgSul: Malto: Tobra IV 0.30 ± 0.10 0.58 ± 0.03 1.93 1.09 1.17 1.29CaSul: Malto: Cipro V 0.28 ± 0.00 0.78 ± 0.02 2.79 1.00 1.00 1.06MgLact: Leu

Table 3 also shows that all five formulations have a HELOS/RODOSdispersibility ratio at 1/4 bar between 1.00 and 1.09, at 0.5/4 barbetween 1.00 and 1.17, and at 0.2/4 bar between 1.06 and 1.29. Valuesthat are close to 1.0, as these values are, are considered indicativethat powders are highly dispersible. In particular, Formulations I and Vdisplayed highly dispersible behavior with all dispersive pressureratios less than 1.1. Specifically, Formulation I contained a very highload of drug (95 percent by weight) with a relatively small amount ofdivalent salt (5 weight percent of magnesium sulfate or 1 weight percentof magnesium ion).

Example 2 Dispersibility of Divalent Cationic Dry Powder

This example demonstrates the dispersibility of dry powder formulationswhen delivered from a dry powder inhaler over a range of inhalation flowrates and volumes.

The dispersibility of various powder formulations was investigated bymeasuring the geometric particle size distribution and the percentage ofpowder emitted from capsules when inhaling on a dry powder inhaler withflow rates representative of patient use. The particle size distributionand weight change of the filled capsules were measured for multiplepowder formulations as a function of flow rate and inhaled volume in apassive dry powder inhaler.

Powder formulations were filled into size 3 HPMC capsules (V-Caps,Capsugel) by hand with the fill weight measured gravimetrically using ananalytical balance (Mettler Toledo X5205). Fill weights of 20 mg werefilled for Formulations I and V. A capsule-based passive dry powderinhaler (RS-01 Model 7, High Resistance, Plastiape S.p.A.) was usedwhich had specific resistance of 0.036 kPa^(1/2)LPM⁻¹. Flow rate andinhaled volume were set using a timer controlled solenoid valve and flowcontrol valve with an inline mass flow meter (TSI model 3063). Capsuleswere placed in the dry powder inhaler, punctured and the inhaler sealedinside a cylinder, exposing the air jet exiting from the DPI to thelaser diffraction particle sizer (Spraytec, Malvern) in its open benchconfiguration. The steady air flow rate through the system was initiatedusing the solenoid valve and the particle size distribution was measuredvia the Spraytec at lkHz for the duration of the single inhalationmaneuver with a minimum of 2 seconds. Particle size distributionparameters calculated included the volume median diameter (Dv50) and thegeometric standard deviation (GSD). At the completion of the inhalationduration, the dry powder inhaler was opened, the capsule removed andre-weighed to calculate the mass of powder that had been emitted fromthe capsule during the inhalation duration. Four inhalation conditionswere used for each powder including 60 LPM and 2 L for the highestinhalation energy condition; 30 LPM and 1 L; 20 LPM and 1 L; and 15 LPMand 1 L for the lowest inhalation energy condition. At each testingcondition, 5 replicate capsules were measured and the results of Dv50,GSD and capsule emitted powder mass (CEPM) were averaged.

In order to relate the dispersion of powders at different flow rates,volumes, and from inhalers of different resistances, the energy requiredto perform the inhalation maneuver was calculated Inhalation energy wascalculated as E=R²Q²V where E is the inhalation energy in Joules, R isthe inhaler resistance in kPa^(1/2)/LPM, Q is the steady flow rate inL/min and V is the inhaled air volume in L. For the example describedhere, the inhalation energy for the case of 60 LPM and 2 L was 9.2Joules while for the lowest case of 15 LPM and 1 L, the inhalationenergy was 0.29 Joules.

Table 4 shows the dose emitted from a capsule (CEPM), and the particlesize distribution parameters of the power emitted (Dv50 and GSD) forFormulations I and V at a capsule fill weight of 20 mg using the highresistance RS-01 dry powder inhaler. For Formulation V, the CEPMremained primarily unchanged as a function of decreased inhalationenergy while the Dv50 increased only slightly from 2.33 to 3.24micrometers, demonstrating excellent flow rate independence of both theamount of powder output and the size of the powder that exited the DPI.For Formulation I, while the CEPM decreased with decreasing inhalationenergy, at least 25% of the filled dose is able to be emitted even downto the very low inhalation energies tested. The Dv50 increased withdecreasing inhalation energy, but the Dv50 remained below 6 micrometersfor all tested conditions.

TABLE 4 Dispersibility of divalent cationic dry powders. Flow Rate:(LPM) 60 30 20 15 Formulation I Dv(50) (μm): 1.48 ± 0.06 1.77 ± 0.062.35 ± 0.32 5.31 ± 2.62 MgSul:Albu GSD (μm): 3.71 ± 0.46 4.27 ± 1.075.19 ± 1.82 7.63 ± 1.00 CEPM (%): 73% 44% 25% 33% Formulation V Dv(50)(μm): 2.33 ± 0.17 2.14 ± 0.15 2.69 ± 0.15 3.24 ± 0.12 MgLact:Leu GSD(μm): 6.26 ± 0.40 4.28 ± 0.65 3.53 ± 0.57 2.55 ± 0.41 CEPM (%): 94% 93%80% 93%

Example 3 Aerodynamic Particle Size

This example demonstrates that the aerodynamic size distribution of drypowder formulations comprised in part of divalent cationic salts, whendelivered from a dry powder inhaler, is in a range appropriate fordeposition in the respiratory tract.

The aerodynamic particle size distributions of five powder formulationswere measured by characterizing the powders with an eight stage Andersoncascade impactor (ACI). Powder formulations were filled into size 3 HPMCcapsules (V-Caps, Capsugel) by hand with the fill weight measuredgravimetrically using an analytical balance (Mettler Toledo XS205). Fillweights of 20 mg were filled for Formulations I and V, and fill weightsof 50 mg were filled for Formulations II, III and IV. A reloadable,capsule-based passive dry powder inhaler (RS-01 Model 7, HighResistance, Plastiape, Osnago, Italy) was used to disperse the powderinto the cascade impactor. One capsule was used for each measurement,with two actuations of 2 L of air at 60 LPM drawn through the dry powderinhaler (DPI). The flow rate and inhaled volume were set using a timercontrolled solenoid valve with flow control valve (TPK2000, CopleyScientific). Three replicate ACI measurements were performed for eachformulation. The impactor stage plates were inverted and pre-weighed 81mm glass fiber filters (1820-6537, Whatman) were placed on them. Afterthe inhalation maneuver, the impactor was disassembled and the glassfiber filters were weighed to determine the mass of powder deposited oneach stage and on the final filter. The size distribution of the emittedpowder was averaged across the replicates and the average mass of powderdelivered to each of the stages (−1, −0, 1, 2, 3, 4, 5, 6, and F) areshown for each formulation in FIGS. 1A to 1E with error bars giving thestandard deviation of the 3 replicates. The mass median aerodynamicdiameter (MMAD), geometric standard deviation (GSD), and fine particledose (FPD<4.4 μm) of the emitted powder were calculated and averagedacross the replicates and the tabulation is shown in Table 5.

All five formulations were found to have repeatable size distributionsas illustrated by the low standard deviations for all the stages andcalculated values. All five formulations had respirable sizedistributions with all five formulations having MMADs of less than 5micrometers. With a maximum GSD of 1.88 for the five formulations, thepolydispersity of the size distributions was relatively small comparedto typical dry powder formulations for inhalation. The fine particledose shown in Table 5 for the five powder formulations demonstrated thata significant mass of the powder dose was contained in small diameterparticles, listed here as less than 4.4 micrometers in diameter; dryparticles of this size would be expected to deposit in the lung. Table 5further demonstrates the delivery of fine particle doses of up to 24 mgof drug formulation from a single size 3 capsule.

TABLE 5 Aerodynamic particle size of divalent cationic dry powders. aPSD(ACI-8) Formulation FPD < 4.4 Number MMAD (μm) GSD (μm) μm (mg) I 2.52 ±0.16 1.83 ± 0.04  8.9 ± 2.1 MgSul:Albu II 2.80 ± 0.10 1.88 ± 0.01 16.3 ±5.7 CaSul:Malto:Cipro III 2.96 ± 0.07 1.84 ± 0.01 23.9 ± 0.7MgSul:Malto:Tobra IV 2.75 ± 0.07 1.87 ± 0.01 19.3 ± 0.3CaSul:Malto:Cipro V 3.56 ± 0.02 1.82 ± 0.01 10.1 ± 0.2 MgLact:Leu

Table 6 lists multiple calcium and magnesium salts. The table showstheir chemical formula, the molecular weights (MW) of each salt, and therelative percentage of the divalent cation (e.g., Ca²⁺, Mg²⁺) in thesalt. The divalent cation can comprise a large, relative percentage ofthe weight of the salt either because of the divalent cation's relativeweight in comparison to the other components of the salt, e.g., calciumor magnesium chloride and/or the salt contains multiple of the divalentcation, e.g., calcium or magnesium citrate.

TABLE 6 Weight Percent of Ca²⁺ in Salt Molecules Weight % of Calcium ionin Salt Molecule Weight % of Ca²⁺ MW in Salt Formula (g/mol) moleculeCalcium carbonate CaCO₃ 100.09 40.0 Calcium chloride CaCl₂ 110.98 36.0Calcium phosphate CaHPO₄ 136.06 29.4 dibasic Calcium sulfate CaSO₄136.14 29.4 Calcium acetate Ca(C₂H₃O₂)₂ 158.17 25.3 Calcium citrateCa₃(C₆H₅O₇)₂ 498.46 24.1 Calcium lactate Ca(C₃H₅O₃)₂ 218.218 18.3Calcium sorbate CaC₁₂H₁₄O₄ 262.33 15.2 Calcium gluconate CaC₁₂H₂₂O₁₄430.373 9.3 Calcium stearate CaC₃₆H₇₀O₄ 607.02 6.6 Calcium alginate[Ca(C₆H₇O₆)₂]_(n) NA NA Weight % of Magnesium ion in Salt MoleculeWeight % of Mg²⁺ MW in Salt Formula (g/mol) molecule Magnesium carbonateMgCO₃ 84.31 28.8 Magnesium carbonate (MgCO₃)₄•Mg(OH)₂ 395.61 30.7hydroxide Magnesium chloride MgCl₂ 95.21 25.5 Magnesium citrateMg₃(C₆H₅O₇)₂ 451.11 16.2 tribasic Magnesium lactate Mg(C₃H₅O₃)₂ 202.4512.0 Magnesium sulfate MgSO₄ 120.37 20.2 Weight % of Monovalent ion inSalt Molecule Weight % of cation Molecular MW in Salt Formula (g/mol)molecule Potassium chloride KCl 74.55 52.4 Potassium citrate C₆H₅K₃O₇306.39 38.3 Sodium ascorbate C₆H₇NaO₆ 198.11 11.6 Sodium bicarbonateCHNaO₃ 84.01 27.4 Sodium carbonate CNa₂O₃ 105.99 43.4 Sodium chlorideNaCl 58.44 39.3 Sodium citrate C₆H₅Na₃O₇ 258.07 26.7 Sodium lactateC₃H₅NaO₃ 112.06 20.5 Dibasic sodium phosphate HNa₂O₄P 141.96 32.4 Sodiumpropionate C₃H₅NaO₂ 96.06 23.9 Sodium sulfate Na₂O₄S 142.04 32.4

Example 4 Magnesium Salt-Containing Dry Powders, Optionally Combinedwith Active Pharmaceutical Agents

A. Powder Preparation.

Feedstock solutions were prepared in order to manufacture dry powderscomprised of dry particles containing a magnesium salt, a non-saltexcipient, and optionally, at least one pharmaceutical active agent.Table 7 lists the components of the feedstock formulations used inpreparation of the dry powders comprised of dry particles. Weightpercentages are given on a dry basis.

TABLE 7 Feedstock compositions of magnesium-salt with excipient, andoptionally, with a pharmaceutically active agents % Salt % Excipient %Drug load load load Formulation Salt (w/w) Excipient (w/w) Drug (w/w) VIMagnesium 9 Maltodextrin 90.9 Tiotropium .113 lactate bromide (TioB) VIIMagnesium 10 Mannitol 90 N/A N/A lactate VIII Magnesium 10 Maltodextrin90 N/A N/A lactate IX Magnesium 10 Leucine 90 N/A N/A sulfate XMagnesium 10 Leucine 90 N/A N/A lactate N/A = not applicable

The feedstock solutions were made according to the parameters in Table8.

TABLE 8 Formulation Conditions Formulation: VI VII VIII IX X Totalsolids (g) 4 3 3 3 3 Total volume water (L) 0.4 0.3 0.3 0.3 0.3 Totalsolids concentration 10 10 10 10 10 (g/L) Amount of magnesium 0.9 1.01.0 1.0 1.0 lactate in 1 L (g) Amount of magnesium 0 0 0 0 0 sulfate in1 L (g) Amount of maltodextrin in 9.09 0 9.0 0 0 1 L (g) Amount ofmannitol in 1 L 0 9.0 0 0 0 (g) Amount of leucine in 1 L 0 0 0 9.0 9.0(g) Amount TioB in 1 L (g) 0.0113 0 0 0 0 For all formulations, theliquid feedstock was batch mixed

Formulation VI through X dry powders were produced by spray drying onthe Büchi B-290 Mini Spray Dryer (BUCHI Labortechnik AG, Flawil,Switzerland) with powder collection from a High Performance cyclone in a60 mL glass vessel. The system used the Büchi B-296 dehumidifier and anexternal LG dehumidifier (model 49007903, LG Electronics, EnglewoodCliffs, N.J.) was run constantly. Atomization of the liquid feedutilized a Büchi two-fluid nozzle with a 1.5 mm diameter. The two-fluidatomizing gas was set at 40 mm (667 LPH) and the aspirator rate to 80%(32 cubic meters per hour). Air was used as the drying gas and theatomization gas. Table 9 below includes details about the spray dryingconditions.

TABLE 9 Spray Drying Process Conditions Formulation Process ParametersVI VII VIII IX X Liquid feedstock solids concentration 10 10 10 10 10(g/L) Process gas inlet temperature (° C.) 100 100 100 115 115 Processgas outlet temperature (° C.) 54-59 55-56 56-60 63-66 65 Process gasflowrate (liter/hr, LPH) 667 667 667 667 667 Atomization gas flowrate(meters³/hr) 32 32 32 32 32 Liquid feedstock flowrate (mL/min) 2.8 2.62.6 2.5 2.6

B. Powder Characterization.

Powder physical and aerosol properties are summarized in Tables 10 to 14below. Values with ±indicate standard deviation of the value reported.Two-stage ACI-2 results are reported in Table 10 for FPF_(TD)<3.4 μm andFPF_(TD)<5.6 μm. Formulations VI, VIII, IX and X all had a FPF_(TD)<3.4μm greater than 20%. Formulations VIII, IX and X had a FPF_(TD)<3.4 μmgreater than 30%. Formulations VI, VIII, IX and X all had a FPF_(TD)<5.6μm greater than 35%. Formulations VIII, IX and X had a FPF_(TD)<5.6 μmgreater than 55%.

TABLE 10 Aerodynamic properties ACI-2 FPF_(TD) < 3.4 μm FPF_(TD)< 5.6 μmFormulation % % VI 22.99% ± 0.12% 37.99% ± 2.00% VII 14.05% ± 0.88%26.72% ± 1.32% VIII 33.67% ± 5.83% 57.57% ± 5.31% IX 40.46% ± 3.54%69.63% ± 4.02% X 46.45% ± 0.99% 71.99% ± 0.90%

Density-related measurements are listed in Table 11. Formulations VI,VII and VIII had a tapped density greater than 0.60 g/cc. Allformulations had a Hausner Ratio greater than 1.4. Formulations VII,VIII, IX and X had Hausner Ratios greater than 2.2.

TABLE 11 Density properties of the magnesium salts Density Bulk TappedHausner Formulation g/cc g/cc Ratio VI 0.44 ± 0.04 0.64 ± 0.01 1.46 VII0.32 ± 0.04 0.79 ± 0.09 2.47 VIII 0.30 ± 0.01 0.75 ± 0.11 2.52 IX 0.11 ±0.00 0.35 ± 0.03 3.17 X 0.14 ± 0.04 0.33 ± 0.06 2.34

Table 12 shows that formulations VIII, IX and X had geometic diameters(Dv50) of less than 2.2 microns when emitted from a dry powder inhalerat a flowrate of 60 LPM. Formulations IX and X had a Dv50 just under 2.0microns. Formulations VIII, IX and X had a Dv50 of less than 6.5 micronsat 15 LPM. Formulation VIII had a Dv50 at 15 LPM of 2.47 microns.

TABLE 12 Geometric Diameters Dispersibility - Spraytec @ 60 LPM @ 15 LPMFormulation Dv50 (μm) GSD Dv50 (μm) GSD VII 6.83 ± 0.63 3.47 ± 0.1117.32 ± 6.99  4.63 ± 0.50 VIII 2.17 ± 0.05 5.91 ± 0.16 2.47 ± 0.14 3.10± 0.64 IX 1.98 ± 0.06 3.62 ± 0.06 5.64 ± 0.36 3.04 ± 0.13 X 1.99 ± 0.093.66 ± 0.20 6.05 ± 0.15 3.05 ± 0.16

Table 13 shows that all formulations had a capsule emitted particle mass(CEPM) of greater than 97% at 60 LPM. All formulations had a CEPM ofgreater than 80% at 15 LPM. Formulations XXI, XXII, and XXIII each had aCEPM of greater than 92% at 15 LPM. Formulations XXII and XXIII each hada CEPM of greater than 97% at 15 LPM. Formulation VI was not tested forDv50 or CEPM across varing inspiratory flow rate.

TABLE 13 Dispersitibilty properties Dispersibility - CEPM @ 60 LPM @ 15LPM Formulation CEPM CEPM VII 99.13% ± 0.29%  87.79% ± 17.84% VIII97.54% ± 0.56% 94.33% ± 0.34% IX 100.24% ± 0.19%  98.97% ± 0.32% X99.22% ± 0.32% 98.33% ± 0.24%

Table 14 shows that all formulations had a Dv50 of about 3.0 microns orless using the RODOS at a 1.0 bar setting. Formulations VI, VIII, IX andX each had a Dv50 of less than 2.4 microns. Formulations IX and X eachhad a Dv50 of about 2.0 microns or less. All measured formulations had aRODOS Ratio for 0.5 bar/4 bar of less than 1.1. All measuredformulations had a RODOS Ratio for 1 bar/4 bar of less than 1.05, withFormulation VII giving an odd result of less than 1.

TABLE 14 Dispersitibilty properties (Geometric diameter using RODOS)RODOS 0.5 bar 1.0 bar 4.0 bar Dv50 Dv50 Dv50 0.5/4 1/4 Form. (μm) GSD(μm) GSD (μm) GSD bar bar VI 2.16 2.09 2.13 2.09 2.08 2.11 1.04 1.02 VII3.67 2.74 3.02 2.60 3.59 2.61 1.02 0.84 VIII 2.41 2.08 2.37 2.09 2.362.08 1.02 1.00 IX 2.09 1.85 1.97 1.81 1.91 1.80 1.09 1.03 X 1.85 1.641.79 1.67 1.74 1.65 1.06 1.03

Example 5 Effect of a Divalent Metal Cation-Based Dry Powder FormulationContaining Tiotropium Bromide on Airway Hyperreactivity in an OvalbuminMouse Model of Allergic Asthma

A mouse model of allergic asthma was established using ovalbumin (OVA).Balb-c mice were sensitized to ovalbumin (OVA) over a period of twoweeks and subsequently challenged via aerosol with OVA as indicated inSchematic 2 below. Sensitizations were performed by intraperotinealinjection of OVA plus Alum. Challenges were performed by whole bodyexposure to nebulized 1% OVA solution for 20 minutes. As presented inTable 15 below, mice were treated with a dry powder comprised of 90.9%maltodextrin, 9% magnesium lactate, 0.113% tiotropium bromide(Formulation VI) or a placebo dry powder of 98% Leucine, 2% sodiumchloride, w/w on a dry basis (Placebo-B). 1 hour before pulmonaryfunction testingon day 30. Treatments were made in a whole body exposurechamber using a capsule based dry powder inhaler system. It was knownfrom the literature that tiotropium bromide (TioB) enhances pulmonaryfunction, resulting in lower sRaw values, for animals and human beingschallenged with methacholine chloride (MCh) in 0.9% sodium chloride forinhalation. Data depict the mean±SEM of 5 mice per group. Data wereanalyzed by Student's t-test, and asterics presented in FIGS. 3A and 3Brepresent a p-value of p<0.05. (Ohta, S. et al. (2010), “Effect oftiotropium bromide on airway inflammation and remodeling in a mousemodel of asthma”, Clinical and Experimental Allergy 40:1266-1275).

TABLE 15 Divalent metal cation salt-based tiotropium dry powderformulation % Salt % Excipient % Drug load load load Formulation Salt(w/w) Excipient (w/w) Drug (w/w) Placebo-B Sodium 2 Leucine 98 N/A N/Achloride VI Magnesium 9 Maltodextrin 90.9 Tiotropium .113 lactatebromide (TioB)

While the effects of TioB on sRaw were known from the literature, theeffect of co-formulating the TioB formulation with a magnesium salt wasunknown. The effect of Formulation VI was tested, and compared toPlacebo-B. Results from pulmonary function testing are shown in FIG XYZ.These data show that Formulation VI significantly reduced sRaw duringMCh challenge compared to Placebo-B (p<0.00001).

The content of each of the patents, patent applications, patentpublications and published articles cited in this specification areherein incorporated by reference in their entirety.

What is claimed is:
 1. A respirable dry powder comprising respirable dryparticles that comprise a) a magnesium salt; wherein the magnesium saltprovides divalent metal cation in an amount between about 0.1% and 2.9%by weight of the dry particle, and b) a pharmaceutical agent, andwherein the respirable dry particles have a volume median geometricdiameter (VMGD) of about 10 microns or less and a dispersibility ratio(1 bar/4 bar) of less than about 1.5 as measured by laser diffraction(RODOS/HELOS system), and wherein the respirable dry particles have atap density of about 0.45 g/cc or greater.
 2. The respirable dry powderof claim 1, wherein the respirable dry powder has a volume mediangeometric diameter (VMGD) of about 5.0 microns or less. 3-4. (canceled)5. The respirable dry powder of claim 1, wherein the respirable drypowder has a Fine Particle Fraction (FPF) of less than 5.6 microns of atleast 45%. 6.-7. (canceled)
 8. The respirable dry powder of claim 1,wherein the respirable dry powder has a mass median aerodynamic diameter(MMAD) of about 5 microns or less.
 9. The respirable dry powder of claim1, wherein the magnesium salt has a solubility of ≧0.5 μg/L in water.10. (canceled)
 11. The respirable dry powder of claim 1, wherein themolecular weight ratio of divalent metal cation to the divalent metalcation salt is greater than about 0.1.
 12. (canceled)
 13. The respirabledry powder of claim 1, wherein the respirable dry powder furthercomprise at least one pharmaceutically acceptable excipient.
 14. Therespirable dry powder of claim 13, wherein the at least one excipient ispresent in an amount of about ≦20% by weight and comprises leucine.15.-16. (canceled)
 17. The respirable dry powder of claim 13, whereinthe at least one excipient is present in an amount of about ≦20% byweight and comprises maltodextrin or mannitol. 18.-21. (canceled)
 22. Arespirable dry powder comprising respirable dry particles that comprisea) a calcium salt; wherein the calcium salt provides divalent metalcation in an amount between about 0.1% and 2.9% by weight of the dryparticle, and b) a pharmaceutical agent, and wherein the respirable dryparticles have a volume median geometric diameter (VMGD) of about 10microns or less and a dispersibility ratio (1 bar/4 bar) of less thanabout 1.5 as measured by laser diffraction (RODOS/HELOS system), andwherein the respirable dry articles have a tap density of about 0.45g/cc or greater.
 23. (canceled)
 24. The respirable dry powder of claim22, wherein the calcium salt is calcium lactate. 25.-34. (canceled) 35.The respirable dry powder of claim 1, wherein the pharmaceuticallyactive agent is an antibiotic, a LABA, a LAMA, a corticosteroid, or anycombination thereof.
 36. The respirable dry powder of claim 1, whereinthe pharmaceutically active agent is a macromolecule.
 37. The respirabledry powder of claim 1, wherein the pharmaceutically active agent is anantibody. 38.-64. (canceled)
 65. A method for treating a respiratorydisease comprising administering to the respiratory tract of a patientin need thereof an effective amount of a respirable dry powder of claim1, wherein the respiratory disease is asthma, airwayhyperresponsiveness, seasonal allergic allergy, bronchiectasis, chronicbronchitis, emphysema, chronic obstructive pulmonary disease, or cysticfibrosis.
 66. A method for treating or preventing an acute exacerbationof a respiratory disease comprising administering to the respiratorytract of a patient in need thereof an effective amount of a respirabledry powder of claim 1, wherein the respiratory disease is asthma, airwayhyperresponsiveness, seasonal allergic allergy, bronchiectasis, chronicbronchitis, emphysema, chronic obstructive pulmonary disease, or cysticfibrosis.
 67. A method for treating or preventing an infectious diseaseof the respiratory tract comprising administering to the respiratorytract of a patient in need thereof an effective amount of a respirabledry powder of claim
 1. 68.-71. (canceled)
 72. The respirable dry powderof claim 1, wherein the therapeutic agent is an antibiotic.
 73. Therespirable dry powder of claim 72, wherein the antibiotic islevofloxacin.
 74. The respirable dry powder of claim 1, wherein thetherapeutic agent is a MABA.
 75. The respirable dry powder of claim 1,wherein the magnesium salt is magnesium lactate.
 76. The respirable drypowder of claim 1, wherein the respirable dry particles are furthercharacterized by a capsule emitted powder mass (CEPM) of at least 80%when emitted from a passive dry powder inhaler under the followingconditions: a total inhalation energy of less than about 2 Joules usinga size 3 capsule that consists of 50 mg of the respirable dry particles.77. The respirable dry powder of claim 1, wherein the respirable dryparticles are further characterized a geometric size (Dv50) of below 6micrometers when emitted from a passive dry powder inhaler that has aresistance of about 0.036 sqrt(kPa)/liters per minute (LPM) under thefollowing conditions: an inhalation flowrate of 30 LPM, an inhalationvolume of 1 liter using a size 3 capsule that contains a total mass of20 mg, said total mass consisting of the respirable dry particles.